Dissertations / Theses: 'Protein flexibility'

1

Mitchell, Felicity. "Modelling protein flexibility using molecular simulation methods." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.525167.

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2

Broadhead, Richard Ian. "Metabolic flexibility in Escherichia coli." Thesis, University of Aberdeen, 1998. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU104201.

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This study of the relationship between heterologous protein production and bacterial growth has shown that foreign protein can account for as much as 30% of the total cell protein without the specific growth rate being affected. Higher levels of foreign protein production are achieved at lower growth rates. Foreign protein is synthesised without any increase in ribosome number or ribosome activity occurring in the recombinant cells relative to their parent strain, i.e. the capacity of parental and recombinant cells to synthesise protein is the same. These observations are explained in terms of a model in which Escherichia coli proteins are be divided into two types. Type I proteins are involved in the processes of transcription and translation. Type 2 proteins are those which serve other purposes. The model suggests that the synthesis of type 2 proteins will be decreased in order to accommodate foreign protein production. This implies that some Escherichia coli proteins are overproduced relative to the amount of that protein that is essential to the cell to maintain growth. Two dimensional electrophoresis showed that there was a decrease in some host cell protein levels between parental and recombinant cells. An increase in the levels of molecular chaperone proteins in recombinant cells grown under some conditions was observed. These data are explained in terms of our current understanding of the regulation of molecular chaperone production. The data obtained are discussed in relation to our current understanding of the growth of bacteria and the reported effects of high levels of foreign protein production on bacterial physiology.

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Magnusson, Ulrika. "Structural Studies of Binding Proteins: Investigations of Flexibility, Specificity and Stability." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3640.

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4

Zöllner, Frank G. "Enhancing protein-protein docking by new approaches to protein flexibility and scoring of docking hypotheses." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972854142.

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5

Dobbins, Sara E. "Computational Studies of Protein Flexibility using Normal Mode Analysis." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508940.

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6

Koch, Kerstin. "Statistical analysis of amino acid side chain flexibility for 1:n protein protein docking." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968919413.

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7

Sánchez, Martínez Melchor. "Protein Flexibility: From local to global motions. A computational study." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/288044.

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Proteins are flexible entities, and thus move. Its function is closely related to the flexibility. To carry out any function is necessary a conformational change. As protein motions imply an exchange of conformations, protein dynamics is also known as Protein Conformational Dynamics. The fluctuations between the different proteic configurations can be classified according to the length-scale, the time-scale and the amplitude and directionality of them. The movement could be local movement, involving only the rearrangement of a few amino- acid side chains or even backbone, or it may be a large, global movement, modulating the allostery or the conformational transitions, and even involve folding of the entire protein. Generally local motions are also fast and small amplitude motions whereas global motions are associated with slow and large amplitude motions. All these motions encompassed into protein dynamics are governed by the features of the underlying energy landscape. To fully describe a protein, a multidimensional and rugged energy landscape defining the relative probabilities of the conformational states (thermodynamics) and the energy barriers between them (kinetics) is required. To understand proteins in action, the fourth dimension, time, must be added. As these motions are important for the proteic function a deep understanding is required. To do that in this thesis we have tried to answer several questions related to these dynamical phenomena. Concretely we have performed local studies, related to enzyme catalysis and protein damage, and globally, with the determination and analysis of protein conformational ensembles. These studies have been developed using methods of computational chemistry, computational biochemistry and computational biophysics, which have proven to be very useful tools when studying the protein dynamics. Computational methods are an efficient and useful tool to characterize protein motions. However the current computational approaches present limitations and to solve them the incorporation of experimental data and its correct interpretation is crucial. This necessity of be complemented comes from different sides: 1) from experiments to computations and 2) from computations to experiments. The convergence of experimental and computational techniques to the same point is key to achieve a deep understanding of protein dynamics.
La presente tesis se centra en el estudio computacional de la dinámica de las proteínas. Las proteínas son entidades flexibles y como tales se mueven. Este movimiento es indispensable y esta directamente relacionado con su función. La dinámica de las proteínas se puede dividir en dos grandes bloques conceptuales según el número de átomos involucrados, la escala de tiempo en que tiene lugar y la amplitud y dirección de la misma. Debido a la importancia de estos fenómenos, emerge la necesidad de tener un conocimiento profundo sobre los mismos. Debido a ello, en esta tesis doctoral se ha tratado de dar respuesta a varios fenómenos observados en relación directa con la dinámica de las proteínas. Concretamente, hemos realizado estudios a nivel local, de 'centro activo', relacionados con la catálisis enzimática y el daño proteico, así como a nivel global, con la determinación y el análisis de conjuntos conformacionales de proteínas. Estos estudios, se han desarrollado usando métodos propios de la química, la bioquímica y la biofísica computacionales, los cuales se han mostrado como herramientas muy útiles a la hora de estudiar la dinámica. De todos ellos, de forma general, podemos concluir que los métodos computacionales son una herramienta eficaz y util para caracterizar la dinámica de las proteínas. Sin embargo, los métodos computacionales actuales presentan limitaciones y para resolverlos la incorporación de datos experimentales as como su correcta interpretación es crucial. Pero aunque los metodos computacionales necesitan de los experimentales, esta necesidad también se da de manera opuesta. La convergencia de los métodos experimentales y computacionales es clave para poder profundizar en el conocimiento de la dinámica de las proteínas.

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8

Salmaso, Veronica. "Exploring protein flexibility during docking to investigate ligand-target recognition." Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3421817.

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Ligand-protein binding models have experienced an evolution during time: from the lock-key model to induced-fit and conformational selection, the role of protein flexibility has become more and more relevant. Understanding binding mechanism is of great importance in drug-discovery, because it could help to rationalize the activity of known binders and to optimize them. The application of computational techniques to drug-discovery has been reported since the 1980s, with the advent computer-aided drug design. During the years several techniques have been developed to address the protein flexibility issue. The present work proposes a strategy to consider protein structure variability in molecular docking, through a ligand-based/structure-based integrated approach and through the development of a fully automatic cross-docking benchmark pipeline. Moreover, a full exploration of protein flexibility during the binding process is proposed through the Supervised Molecular Dynamics. The application of a tabu-like algorithm to classical molecular dynamics accelerates the binding process from the micro-millisecond to the nanosecond timescales. In the present work, an implementation of this algorithm has been performed to study peptide-protein recognition processes.
I modelli di riconoscimento ligando-proteina si sono evoluti nel corso degli anni: dal modello chiave-serratura a quello di fit-indotto e selezione conformazionale, il ruolo della flessibilità proteica è diventato via via più importante. Capire il meccanismo di riconoscimento è di grande importanza nella progettazione di nuovi farmaci, perchè può dare la possibilità di razionalizzare l’attività di ligandi noti e di ottimizzarli. L’applicazione di tecniche computazionali alla scoperta di nuovi farmaci risale agli anni ‘80, con l’avvento del cosiddetto “Computer-Aided Drug Design”, o, tradotto, progettazione di farmaci aiutata dal computer. Negli anni sono state sviluppate molte tecniche che hanno affrontato il problema della flessibilità proteica. Questo lavoro propone una strategia per considerare la variabilità delle strutture proteiche nel docking, attraverso un approccio combinato ligand-based/structure-based e attraverso lo sviluppo di una procedura completamente automatizzata di docking incrociato. In aggiunta, viene proposta una piena esplorazione della flessibilità proteica durante il processo di legame attraverso la Dinamica Molecolare Supervisionata. L’applicazione di un algoritmo simil-tabu alla dinamica molecolare classica accelera il processo di riconoscimento dalla scala dei micro-millisecondi a quella dei nanosecondi. Nel presente lavoro è stata fatta un’implementazione di questa algoritmica per studiare il processo di riconoscimento peptide-proteina.

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9

Cobb, Andrew Martin. "Resolving the flexibility and intricacy of DNA repair protein-DNA interactions." Thesis, University of East Anglia, 2010. https://ueaeprints.uea.ac.uk/10586/.

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Within all cells, complex molecular systems exist that are responsible for maintaining genome stability by detecting and repairing dangerous alterations in DNA. Ensuring the accurate and efficient functioning of such systems is necessary for the preservation of DNA integrity and avoidance of disease. The flexible and diverse modes of DNA-binding exhibited by human p53 permits this ‘guardian of the genome’ to elicit versatile cellular activities that are crucial in monitoring threats to genome dynamics and conducting appropriate responses. In conjunction with its sequence-specific DNA-binding activity that is essential to target gene transactivation, p53 can bind to unusual DNA structures independent of DNA sequence and it has been proposed this activity may allow p53 to interact with detrimental secondary structures that arise in unstable genomic regions. To provide further insight into p53-DNA interactions, an in vitro DNA binding assay was developed that was used to characterise binding properties towards several DNA molecules to allow comparison of non-specific, sequence-specific and structurespecific binding. It was determined that unusual structures in DNA significantly enhanced p53 binding in non-sequence specific DNA and that the presence of internal hairpin regions induced binding comparable to sequence-specific binding. In vivo p53-DNA interactions were also quantified using chromatin immunoprecipitation and variations in preference to different response element sequences was ascertained. DNA binding is also central to the ability of Ku proteins to function as essential components of non-homologous end joining and telomere maintenance in eukaryotes. Prokaryotic homologues of Ku proteins that function as homodimers in two-component repair systems have also been identified. Recently, 3 Ku homologues in Streptomyces coelicolor were reported, but very little is currently known regarding their biological activity. It was discovered that all 3 Ku proteins exhibited varied independent DNA-binding properties that were influenced by DNA topology, size and end-structure. Unusually for Ku, it was found 1 of these proteins exhibited strong binding to single-stranded DNA. Precipitation assays determined that these proteins may act as DNA end synapsis mediators during the DNA endjoining process and ligation experiments revealed Ku was responsible for rigidifying DNAs or completely inhibiting ligation activity, probably via DNA end-protection activity. Experimental evidence indicated that specific interactions could occur between S. coelicolor Ku suggesting these proteins form both homodimers and heterodimers.

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10

Yu, Hongtao. "Water in Protein Cavities: Free Energy, Entropy, Enthalpy, and its Influences on Protein Structure and Flexibility." ScholarWorks@UNO, 2011. http://scholarworks.uno.edu/td/341.

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Complexes of the antibiotics novobiocin and clorobiocin with DNA gyrase are illustrative of the importance of bound water to binding thermodynamics. Mutants resistantto novobiocin as well as those with a decreased affinity for novobiocin over clorobiocinboth involve a less favorable entropy of binding, which more than compensates for amore favorable enthalpy, and additional water molecules at the proteinligandinterface.Free energy, enthalpy, and entropy for these water molecules were calculated by thermodynamicintegration computer simulations. The calculations show that addition of thewater molecules is entropically unfavorable, with values that are comparable to the measuredentropy differences. The free energies and entropies correlate with the change inthe number of hydrogen bonds due to the addition of water molecules.To examine the wide variety of cavities available to water molecules inside proteins,a model of the protein cavities is developed with the local environment treated at atomicdetail and the nonlocal environment treated approximately. The cavities are then changedto vary in size and in the number of hydrogen bonds available to a water molecule insidethe cavity. The free energy, entropy, and enthalpy change for the transfer of a watermolecule to the cavity from the bulk liquid is calculated from thermodynamic integration.The results of the model are close to those of similar cavities calculated using the fullprotein and solvent environment. As the number of hydrogen bonds resulting from theaddition of the water molecule increases, the free energy decreases, as the enthalpic gainof making a hydrogen bond outweighs the entropic cost. Changing the volume of thecavity has a smaller effect on the thermodynamics. Once the hydrogen bond contributionis taken into account, the volume dependence on free energy, entropy, and enthalpy issmall and roughly the same for a hydrophobic cavity as a hydrophilic cavity.The influences of bound water on protein structure and influences are also evaluatedby performing molecular dynamics simulation for proteins with and without boundwater. Four proteins are simulated, the wildtypebovine pancreatic trypsin inhibitor(BPTI), the wildtypehen egg white lysozyme (HEWL), and two variants of the wildtypeStaphylococcal nuclease (SNase), PHS and PHS/V66E. The simulation reveals that allthese four proteins suffer structural changes upon the removing of bound water molecules,as indicating by their increased RMSD values with respect to the crystal structures. Threeout of the four proteins, BPTI, HEWL, and the PHS mutant of SNase have increased flexibility,while no apparent flexibility change is seen in the PHS/V66E variant of SNase.

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11

Apgar, James R. (James Reasoner). "Modeling the flexibility of alpha helices in protein interfaces : structure based design and prediction of helix-mediated protein-protein interactions." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/43778.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.
Vita.
Includes bibliographical references.
Protein-protein interactions play an essential role in many biological functions. Prediction and design of these interactions using computational methods requires models that can be used to efficiently sample structural variation. This thesis identifies methods that can be used to sample an important sub-space of protein structure: alpha helices that participate in protein interfaces. Helices, the global structural properties of which can be described with only a few variables, are particularly well suited for efficient sampling. Two methods for sampling helical backbones are presented: Crick parameterization for coiled coils and normal-mode analysis for all helices. These are shown to capture most of the variation seen in the PDB. In addition, these methods are applied to problems in protein structure prediction and design. Normal-mode analysis is used to design novel nanomolar peptide inhibitors of the apoptosis-related Bcl-2 family member, Bcl-xL, and a modification of Crick Parameterization is used to predict the binding orientation of dimeric coiled coils with greater than 80% accuracy. Finally, this study addresses the increase in computational time required by flexible-backbone methods and the use of cluster expansion to quickly map structural energies to sequence-based functions for increased efficiency.
by James R. Apgar.
Ph.D.

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12

Regmi, Chola K. "Structural Flexibility and Oxygen Diffusion Pathways in Monomeric Fluorescent Proteins." FIU Digital Commons, 2014. http://digitalcommons.fiu.edu/etd/1122.

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Fluorescent proteins are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. We used molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variants, and its GFP homolog citrine. We employed implicit ligand sampling and locally enhanced sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. The pathway contains several oxygen hosting pockets, which were identified by the amino acid residues that form the pocket. We calculated the free-energy of an oxygen molecule at points along the path. We also investigated an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. We showed that oxygen pathways can be blocked or altered, and barrel fluctuations can be reduced by strategic amino acid substitutions. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins.

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13

Crevenna, Escobar Alvaro Hernan. "Kinesin-1 mechanical flexibility and motor cooperation." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2007. http://nbn-resolving.de/urn:nbn:de:swb:14-1193308684832-88632.

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Conventional kinesin (kinesin-1) transports membrane-bounded cargos such as mitochondria and vesicles along microtubules. In vivo it is likely that several kinesins move a single organelle and it is important that they operate in a coordinated fashion so that they do not interfere with each other. Evidence for coordination comes from in vitro assays, which show that the gliding speed of a microtubule driven by many kinesins is as high as one driven by just a single kinesin molecule. Coordination is thought to be facilitated by flexible domains so that when one motor is bound another can work irrespectively of their orientations. The tail of kinesin-1 is predicted to be composed of a coiled-coil with two main breaks, the “swivel” (380-442 Dm numbering) and the hinge (560-624). The rotational Brownian motion of microtubules attached to a glass surface by single kinesin molecules was analyzed and measured the torsion elasticity constant. The deletion of the hinge and subsequent tail domains increase the stiffness of the motor (8±1 kBT/rad) compared to the full length (0.06±0.01 kBT/rad measured previously), but does not impair motor cooperation (700±16nm/s vs. full length 756±55nm/s - speed in high motor density motility assays). Removal of the swivel domain generates a stiff construct (7±1 kBT/rad), which is fully functional at single molecule (657±63nm/s), but it cannot work in large numbers (151±46nm/s). Due to the similar value of flexibility for both short construct (8±11 kBT/rad vs 7±1 1 kBT/rad) and their different behavior at high density (700±16 nm/s vs. 151±46 nm/s) a new hypothesis is presented, the swivel might have a strain dependent conformation. Using Circular Dichroism and Fluorescence the secondary structure of this tail region was studied. The central part of the swivel is dimeric α-helical and it is surrounded by random coils, thereby named helix-coil (HC) region. Furthermore, an experimental set-up is developed to exert a torque on individual kinesin molecules using hydrodynamic flow. The results obtained suggest for the first time the possibility that a structural element within the kinesin tail (HC region) has a force-dependent conformation and that this allows motor cooperation.

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14

Buonfiglio, Rosa <1985&gt. "Computational strategies to include protein flexibility in Ligand Docking and Virtual Screening." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6330/1/Tesi_Buonfiglio.pdf.

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The dynamic character of proteins strongly influences biomolecular recognition mechanisms. With the development of the main models of ligand recognition (lock-and-key, induced fit, conformational selection theories), the role of protein plasticity has become increasingly relevant. In particular, major structural changes concerning large deviations of protein backbones, and slight movements such as side chain rotations are now carefully considered in drug discovery and development. It is of great interest to identify multiple protein conformations as preliminary step in a screening campaign. Protein flexibility has been widely investigated, in terms of both local and global motions, in two diverse biological systems. On one side, Replica Exchange Molecular Dynamics has been exploited as enhanced sampling method to collect multiple conformations of Lactate Dehydrogenase A (LDHA), an emerging anticancer target. The aim of this project was the development of an Ensemble-based Virtual Screening protocol, in order to find novel potent inhibitors. On the other side, a preliminary study concerning the local flexibility of Opioid Receptors has been carried out through ALiBERO approach, an iterative method based on Elastic Network-Normal Mode Analysis and Monte Carlo sampling. Comparison of the Virtual Screening performances by using single or multiple conformations confirmed that the inclusion of protein flexibility in screening protocols has a positive effect on the probability to early recognize novel or known active compounds.

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Buonfiglio, Rosa <1985&gt. "Computational strategies to include protein flexibility in Ligand Docking and Virtual Screening." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6330/.

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The dynamic character of proteins strongly influences biomolecular recognition mechanisms. With the development of the main models of ligand recognition (lock-and-key, induced fit, conformational selection theories), the role of protein plasticity has become increasingly relevant. In particular, major structural changes concerning large deviations of protein backbones, and slight movements such as side chain rotations are now carefully considered in drug discovery and development. It is of great interest to identify multiple protein conformations as preliminary step in a screening campaign. Protein flexibility has been widely investigated, in terms of both local and global motions, in two diverse biological systems. On one side, Replica Exchange Molecular Dynamics has been exploited as enhanced sampling method to collect multiple conformations of Lactate Dehydrogenase A (LDHA), an emerging anticancer target. The aim of this project was the development of an Ensemble-based Virtual Screening protocol, in order to find novel potent inhibitors. On the other side, a preliminary study concerning the local flexibility of Opioid Receptors has been carried out through ALiBERO approach, an iterative method based on Elastic Network-Normal Mode Analysis and Monte Carlo sampling. Comparison of the Virtual Screening performances by using single or multiple conformations confirmed that the inclusion of protein flexibility in screening protocols has a positive effect on the probability to early recognize novel or known active compounds.

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16

Fischer, Nina M. [Verfasser], and Oliver [Akademischer Betreuer] Kohlbacher. "Modeling Flexibility of Protein-DNA and Protein-Ligand Complexes using Molecular Dynamics / Nina M. Fischer ; Betreuer: Oliver Kohlbacher." Tübingen : Universitätsbibliothek Tübingen, 2013. http://d-nb.info/1162896728/34.

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17

Crevenna, Escobar Alvaro Hernan. "Kinesin-1 mechanical flexibility and motor cooperation." Doctoral thesis, Technische Universität Dresden, 2006. https://tud.qucosa.de/id/qucosa%3A23868.

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Conventional kinesin (kinesin-1) transports membrane-bounded cargos such as mitochondria and vesicles along microtubules. In vivo it is likely that several kinesins move a single organelle and it is important that they operate in a coordinated fashion so that they do not interfere with each other. Evidence for coordination comes from in vitro assays, which show that the gliding speed of a microtubule driven by many kinesins is as high as one driven by just a single kinesin molecule. Coordination is thought to be facilitated by flexible domains so that when one motor is bound another can work irrespectively of their orientations. The tail of kinesin-1 is predicted to be composed of a coiled-coil with two main breaks, the “swivel” (380-442 Dm numbering) and the hinge (560-624). The rotational Brownian motion of microtubules attached to a glass surface by single kinesin molecules was analyzed and measured the torsion elasticity constant. The deletion of the hinge and subsequent tail domains increase the stiffness of the motor (8±1 kBT/rad) compared to the full length (0.06±0.01 kBT/rad measured previously), but does not impair motor cooperation (700±16nm/s vs. full length 756±55nm/s - speed in high motor density motility assays). Removal of the swivel domain generates a stiff construct (7±1 kBT/rad), which is fully functional at single molecule (657±63nm/s), but it cannot work in large numbers (151±46nm/s). Due to the similar value of flexibility for both short construct (8±11 kBT/rad vs 7±1 1 kBT/rad) and their different behavior at high density (700±16 nm/s vs. 151±46 nm/s) a new hypothesis is presented, the swivel might have a strain dependent conformation. Using Circular Dichroism and Fluorescence the secondary structure of this tail region was studied. The central part of the swivel is dimeric α-helical and it is surrounded by random coils, thereby named helix-coil (HC) region. Furthermore, an experimental set-up is developed to exert a torque on individual kinesin molecules using hydrodynamic flow. The results obtained suggest for the first time the possibility that a structural element within the kinesin tail (HC region) has a force-dependent conformation and that this allows motor cooperation.

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18

Sun, Li. "Advances in the use of phosphorescence spectroscopy as a probe of protein flexibility." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq30395.pdf.

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19

Long, Yun Chau. "Skeletal muscle metabolic flexibility: the roles of AMP-activated protein kinase and calcineurin /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-152-4/.

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20

Jaya, Nomalie Naomi. "SUBSTRATE BINDING SITE FLEXIBILITY OF SMALL HEAT SHOCK PROTEINS AND FACTORS CONTRIBUTING TO EFFICIENT CHAPERONE ACTIVITY." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/193550.

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sHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.

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21

Radford, Sheena Elizabeth. "Domains and conformational flexibility in the catalytic mechanism of the 2-oxo acid dehydrogenase complexes." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236036.

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The structure of the dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex from Escherichia coli and its role in catalysis were studied by the combined approaches of protein engineering, limited proteolysis and ¹H-n.m.r. spectroscopy. Genetic reconstruction of the E2p component (performed elsewhere) produced a series of mutant complexes assembled around E2p chains which contain only a single lipoyl domain and an associated (alanine+proline)-rich linker region of gradually diminishing lengths (32, 20, 13, 7 and 1 residue(s), respectively, in the pGS110-,pGS156-,pGS186 ,pGS187- and pGS188-encoded complexes). When this region was shortened to 13 residues or less, the system of active-site coupling in the enzyme complex was dramatically impaired, although the individual enzyme activities were unaffected. The role of the (alanine+proline)-rich region in facilitating moment of the lipoyl domains in catalysis was thus established. The (alanine+proline)-rich regions of the wild-type E2p chains had previously been conjectured to be the source of the unexpectedly sharp resonances in the ¹H-n.m.r. spectrum of the enzyme complex, and hence to be conformationally mobile. Examination of the genetically restructured complexes by ¹H-n.m.r. spectroscopy revealed that the intensity of the sharp peaks in the spectra correlated well with the length of the (alanine+proline)-rich region in each complex. Furthermore, resonances from a single histidine residue engineered into the (alanine+proline)-rich region of a pGS110-encoded E2p chain was clearly visible in the ¹H-n.m.r. spectrum of the resulting enzyme complex. These experiments proved unequivocally that the (alanine+proline)-rich regions are conformationally mobile. The ¹H-n.m.r. spectra of the mutant complexes with the most severe deletions in the E2p chains differed from those of the wild-type and pGS110-encoded complexes in that they displayed a novel sharp peak which was not initially apparent in the spectra of the parent assemblies. This resonance was tentatively assigned to another, shorter (alanine+proline)-rich sequence in the E2p chain, which separates the dihydrolipoamide dehydrogenase (E3)-binding and inner-core domains in the C-terminal half of the molecule. It is likely therefore that this sequence is also conformationally flexible. Antibodies against a synthetic peptide with the sequence of the long (alanine+proline)-rich region of the pGS110-encoded E2p chain were raised elsewhere. Binding of the Fab fragments of these antibodies to the pGS110-encoded complex was found to inhibit the overall complex activity even though the activities of the three component enzymes were not affected. Antibody binding was shown to prevent both the reductive acetylation of the lipoyl domains at the pyruvate decarboxylase (E1p) active site and the transfer of acetyl groups between adjacent lipoyl domains, demonstrating the role of the (alanine+proline)-rich sequence in the mechanism of substrate transfer between active sites. A detailed study of the conformation of the (alanine+proline)-rich regions was also undertaken. Synthetic peptides were obtained with sequences identical to the central and innermost such regions of the wild-type E2p chain. The conformation of these peptides in aqueous solution was studied by circular dichroism, ¹H-n.m.r. and ¹³C-n.m.r. spectroscopy. Relaxation time and nOe data pointed to an extended conformation for the peptides, a structure enforced by the predominantly trans Ala-Pro peptide bond. The functional consequences of this conformation and the role of these sequences in the structure and the function of the enzyme complex are discussed.

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22

Pallara, Chiara. "Structural Modeling and Characterization of Protein Interactions of Biomedical Interest: The Challenge of Molecular Flexibility." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/385987.

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Proteins are large biomolecules that play essential functional and structural roles within cells and that typically act through their interaction with other proteins and biomolecules, forming highly specific functional complexes. Thus, a major biological challenge is to provide structural and energetics details for such interactions. In this context, computational methods can successfully contribute to predict and characterize the mechanistic aspects of protein function, in which conformational flexibility plays a major role. However, an accurate consideration of protein plasticity within computational modeling of protein function at molecular level is still far from trivial, mostly because of both technical and methodological limitations. Thus, the main purpose of this PhD thesis is the assessment, development and application of computational tools for the structural, energetic and dynamic characterization of protein molecules and their interactions. To fulfill these objectives, the first part of the thesis consists in the review and comparison of several existing computational protocols for the characterization of protein-protein interfaces, as well as in the evaluation and discussion of the performance of pyDock, the docking protocol previously developed in our group, as resulted from the last CAPRI (Critical Assessment of PRediction of Interactions) editions. These analyses confirm that current computational protocols aimed to model the phylogenetic, structural and energetic properties of residues within protein-protein interfaces show reasonably good predicting performance and consistency. However, as shown by CAPRI experiment, despite recent methodological advances in docking, dealing with protein plasticity is still a crucial bottle-neck. Based on these premises, the second part of the thesis is focused on understanding the role of protein conformational heterogeneity in protein-protein recognition. Subsequently, a novel protocol to integrate unbound conformational ensembles within a docking framework has been devised and systematically tested. The analysis of conformational heterogeneity in precomputed unbound ensembles reveals that docking encounters are favoured by improving the energetic complementarity of the docking partners rather than the geometrical similarity to the bound state. Moreover, the unbiased use of such ensembles is a successful strategy to incorporate flexibility into a docking approach for low-medium flexible cases, especially those that presumably follow a conformational selection mechanism. Finally the last part of the thesis consists in the application of computational methods to the modeling of protein interactions and the exhaustive exploration of their conformational space within different realistic contexts, thanks to the expertise previously acquired on the theoretical basis of protein interactions. Thus, the main lines of research include the energetic characterization of host-pathogen protein interactions (i.e., host GTPase Rab5 with pathogen phospholipase VipD), the ab-initio modeling of the encounter complex ensembles of redox proteins (i.e. PSI with alternative electron donors, cytochrome c6 and plastocyanin), and finally the description of the structural and dynamic basis of a protein kinase dysfunction under pathological conditions (i.e., MEK1 oncogenic and CFC-related mutants). The results confirmed that computational modeling can complement experimental data to improve the understanding of biological processes involving protein interactions and can help to rationalize and quantify the structural, energetic and dynamic effects of pathological mutations at molecular level.
Las proteínas son grandes biomoléculas que desarrollan funciones esenciales en las células, muy a menudo mediante la formación de complejos altamente específicos con otras proteínas y biomoléculas. Por tanto, uno de los mayores retos científicos en la actualidad es el estudio completo a nivel estructural y energético de todas las interacciones entre proteínas de interés biológico y terapéutico. Sin embargo, la consideración precisa de la plasticidad de las proteínas en los métodos computacionales de modelado molecular no es trivial, debido a limitaciones tanto técnicas como metodológicas. En este contexto, el objetivo principal de esta tesis doctoral ha sido el desarrollo, aplicación y evaluación de herramientas computacionales para la caracterización estructural, energética y dinámica de las proteínas y sus interacciones. Para cumplir con estos objetivos, durante la primera parte de la tesis se ha llevado a cabo la revisión de varios protocolos computacionales para la caracterización de las superficies de interación entre proteínas. Los métodos analizados proporcionan unas predicciones razonablemente consistentes y fiables. También se ha llevado a cabo la evaluación de la eficacia predictiva de nuestro método pyDock en CAPRI, un experimento comunitario de evaluación de métodos de modelado estructural de complejos entre proteínas. En general, a pesar de los avances metodológicos en los protocolos de docking, el modelado eficaz de la plasticidad de las proteínas sigue siendo un reto importante en el campo. En base a los análisis anteriores, se ha llevado a cabo un estudio sistemático sobre la importancia de la heterogeneidad conformacional en el reconocimiento entre proteínas. Los resultados indican que los ensamblados conformacionales generados a partir de proteínas en solución contienen confórmeros con mejor complementariedad energética que la estructura cristalográfica de dichas proteínas y que favorecen su reconocimiento intermolecular. A partir de estos resultados, se ha propuesto un nuevo métodode docking que usa ensamblados conformacionales generados a partir de las proteínas en solución. Esta estrategia resulta particularmente efectiva en casos poco o medianamente flexibles. Finalmente, en la última parte de la tesis se ha llevado a cabo la aplicación de métodos computacionales al modelado de varios casos de interés biomédico. En conclusión, los avances metodológicos en cuanto al modelado de proteínas y sus interacciones, junto a la inclusión eficaz de la flexibilidad conformacional, permiten tener herramientas computacionales cada vez más útiles para complementar los datos experimentales y mejorar la comprensión de procesos biológicos relevantes.

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Kolar, Michal. "Statistical Physics and Message Passing Algorithms. Two Case Studies: MAX-K-SAT Problem and Protein Flexibility." Doctoral thesis, SISSA, 2006. http://hdl.handle.net/20.500.11767/4659.

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In the last decades the tl1eory of spin glasses has been developed within the framework of statistical physics. The obtained results showed to be novel not only from the physical point of vie\l\'1 but they have brought also new mathematical techniques and algorithmic approaches. Indeed, the problem of finding ground state of a spin glass is (in general) NP-complete. The methods that were found brought new ideas to the field of Combinatorial Optimization, and on the other side, the similar methods of Combinatorial Optimization, were applied in physical systems. As it happened with the Monte Carlo sampling and the Simulated Annealing, also the novel Cavity Method lead to algorithms that are open to wide use in various fields of research The Cavity Method shows to be equivalent to Bethe Approximation in its most symmetric version, and the derived algorithm is equivalent to the Belief Propagation, an inference method used widely for example in the field of Pattern Recognition. The Cavity Method in a less symmetric situation, when one has to consider correctly the clustering of the configuration space, lead to a novel messagepassing algorithm-the Survey Propagation. The class of Message-Passing algorithms, among which both the Belief Propagation and the Survey Propagation belong, has found its application as Inference Algorithms in many engineering fields. Among others let us :mention the Low-Density Parity-Check Codes, that are widely used as ErrorCorrecting Codes for communication over noisy cha1mels. In the first part of this work we have compared efficiency of the Survey Propagation Algorithm and of standard heuristic algorithms in the case of the random-MAX-K-SAT problem. The results showed that the algorithms perform similarly in the regions where the clustering of configuration space does not appeai~ but that the Survey Propagation finds much better solutions to the optimization problem in the critical region where one has to consider existence of many ergodic components explicitly. The second part of the thesis targets the problem of protein structure and flexibility. In many proteins the mobility of certain regions and rigidity of other regions of their structure is crucial for their function or interaction with other cellular elements. Our simple model tries to point out the flexible regions from the knowledge of native 3D-structure of the protein. The problem is mapped to a spin glass model which is successfully solved by the Believe Propagation algorithm.

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Munz, Marton. "Computational studies of protein dynamics and dynamic similarity." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:2fb76765-3e43-409b-aad3-b5202f4668b3.

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At the time of writing this thesis, the complete genomes of more than 180 organisms have been sequenced and more than 80000 biological macromolecular structures are available in the Protein Data Bank (PDB). While the number of sequenced genomes and solved three-dimensional structures are rapidly increasing, the functional annotation of protein sequences and structures is a much slower process, mostly because the experimental de-termination of protein function is expensive and time-consuming. A major class of in silico methods used for protein function prediction aim to transfer annotations between proteins based on sequence or structural similarities. These approaches rely on the assumption that homologous proteins of similar primary sequences and three-dimensional structures also have similar functions. While in most cases this assumption appears to be valid, an increasing number of examples show that proteins of highly similar sequences and/or structures can have different biochemical functions. Thus the relationship between the divergence of protein sequence, structure and function is more complex than previously anticipated. On the other hand, there is mounting evidence suggesting that minor changes of the sequences and structures of proteins can cause large differences in their conformational dynamics. As the intrinsic fluctuations of many proteins are key to their biochemical functions, the fact that very similar (almost identical) sequences or structures can have entirely different dynamics might be important for understanding the link between sequence, structure and function. In other words, the dynamic similarity of proteins could often serve as a better indicator of functional similarity than the similarity of their sequences or structures alone. Currently, little is known about how proteins are distributed in the 'dynamics space' and how protein motions depend on structure and sequence. These problems are relevant in the field of protein design, studying protein evolution and to better understand the functional differences of proteins. To address these questions, one needs a precise definition of dynamic similarity, which is not trivial given the complexity of protein motions. This thesis is intended to explore the possibilities of describing the similarity of proteins in the 'dynamics space'. To this end, novel methods of characterizing and comparing protein motions based on molecular dynamics simulation data were introduced. The generally applicable approach was tested on the family of PDZ domains; these small protein-protein interaction domains play key roles in many signalling pathways. The methodology was successfully used to characterize the dynamic dissimilarities of PDZ domains and helped to explain differences of their functional properties (e.g. binding promiscuity) also relevant for drug design studies. The software tools developed to implement the analysis are also introduced in the thesis. Finally, a network analysis study is presented to reveal dynamics-mediated intramolecular signalling pathways in an allosteric PDZ domain.

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25

Tripathi, Swarnendu. "Conformational Transition Mechanisms of Flexible Proteins." Kent State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=kent1281491004.

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26

Mittal, Seema. "Role of Protein Flexibility in Function, Resistance Pathways and Substrate Recognition Specificity in HIV-1 Protease: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/573.

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In the 30 years since the Center for Disease Control's Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens. HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert. Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively. In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable. Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease. This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease.

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Ceres, Nicoletta. "Coarse-grain modeling of proteins : mechanics, dynamics and function." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10030.

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Les protéines sont des molécules flexibles, qui accomplissent une variété de tâches cellulaires à travers des mouvements mécaniques et des changements conformationnels encodés dans leur structure tridimensionnelle. Parmi les approches théoriques qui contribuent à une meilleure compréhension de la relation entre structure, mécanique, dynamique et fonction des protéines, les modèles gros-grains sont un outil très puissant. Ils permettent d’intégrer des informations structurales et dynamiques à un coût computationnel réduit, car le traitement explicite des degrés de liberté moins importants est supprimé. Dans le cadre de cette thèse, des études comparatives rapides de la flexibilité et de la mécanique des protéines ont été menées en se servant du simple modèle gros-grains de Réseau Élastique. La dépendance des résultats de la conformation de départ, ainsi que une liberté dynamique de la chaine principale plutôt limitée, imposée par l’approximation harmonique, nous ont motivé à développer une nouvelle approche, permettant une exploration plus extensive de l’espace conformationnel. Les efforts ont conduit à PaLaCe, modèle gros-grains qui permet des changements majeurs de la structure secondaire, tout en gardant la spécificité de la séquence des acides aminés grâce à une représentation à basse résolution. En utilisant PaLaCe nous avons simulé deux processus impliquant la plasticité protéique: le dépliement du domaine I27 de la protéine musculaire titine et la dynamique à l’équilibre autour de la structure native de deux enzymes homologues adaptées à des températures différentes. Les résultats obtenus concordent avec les données expérimentales et les résultats issus de modèles tout-atom déjà publiés. PaLaCe s’avère donc être un modèle fiable, avec des temps de calcul restreints par rapport aux modèles tout-atome, tout en conservant un bon niveau de détail. Il offre ainsi la possibilité d’effectuer une recherche systématique sur les liens entre mécanique, dynamique et fonction des protéines
Proteins are flexible molecules, which accomplish a variety of cellular tasks through mechanical motions and conformational fluctuations encoded in their three-dimensional structure. Amongst the theoretical approaches contributing to a better understanding of the relationship between protein structure, mechanics, dynamics and function, coarse-grain models are a powerful tool. They can be used to integrate structural and dynamic information over broad time and size scales at a low computational cost, achieved by averaging out the less important degrees of freedom. In this work, fast comparative studies of protein flexibility and mechanics have been performed with the simple coarse-grain Elastic Network Model. However, the dependency of the results on the starting conformation, and the rather constrained backbone dynamics imposed by the harmonic approximation, motivated the development of a new approach, for a more extensive exploration of conformational space. These efforts led to the PaLaCe model, designed to allow significant changes in secondary structure, while maintaining residue specificity despite a lower-level resolution. Using PaLaCe, we were able to reproduce two processes involving protein plasticity: the mechanical unfolding of the I27 domain of the giant muscle protein titin and the near-native dynamics of two homologous enzymes adapted to work at different temperatures. Agreement with experimental data and results from published atomistic models demonstrate that PaLaCe is a reliable, sufficiently accurate, but computationally inexpensive approach. It therefore opens the doors for a systematic investigation of the link between protein dynamics/mechanics and function

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Pondaven, Simon Pierre. "Conformational Flexibility and Amyloid Core Characterization of Human Immunoglobulin Light Chain Domains by Multidimensional NMR Spectroscopy." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354113457.

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SCARAMOZZINO, DOMENICO. "Elastic Lattice Models: From Proteins to Diagrid Tall Buildings." Doctoral thesis, Politecnico di Torino, 2021. http://hdl.handle.net/11583/2872326.

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FRACCALVIERI, DOMENICO. "Comparison of protein dynamics: a new methodology based on self-organizing maps." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/19615.

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The deep relation between dynamics, in terms of both global and local flexibility, and function of proteins is now widely acknowledged. Flexibility is involved in protein binding to small molecules as well as in mechanisms involving domain motions and is also at the basis of signal transduction processes and allosteric interactions. Computational methods, and in particular Molecular-Dynamics (MD) simulations, are widely applied in the investigation of a wide range of dynamic properties and processes that occur in the ps to μs timescale. By means of MD simulations a large ensemble of molecular structures can be generated to sample the accessible conformational space of a protein and identification of functionally relevant conformations is generally done by comparing and grouping the obtained conformations. Data-mining techniques, like clustering, provide one means to group and analyse the information in the MD trajectory, but the results are often influenced by the type of algorithm and the choice of optimal parameters is often case dependent. On the other hands, nowadays computer technology has simplified the complexity in analysing and visualizing scientific data. Within these techniques, the Self-Organizing Maps (SOMs) are an invaluable data mining tool. In this thesis a novel strategy to analyse and compare conformational ensembles of protein domains was developed by using a two-level approach that combines SOMs and complete linkage clustering. First, the representation of the conformations extracted from the MD simulations were encoded as a proper input data for the SOM analysis. Second, the effects of the typical SOM parameters in the analysis of these data were studied by using an experimental design approach. Third the use of a rule to define the optimal number of clusters that best summarizes the information in the map was proposed. Finally this new protocol was applied for the conformational and functional analysis of two study cases: a) a group of single-site mutants of the α-spectrin SH3 (Spc-SH3) domain and b) the bound and unbound states of a group of protein-protein complexes involving proteins of the RAS superfamily. The results demonstrated the potential of this approach in the analysis of large ensembles of molecular conformations, i.e. the possibility of producing a topological mapping of the conformational space in a simple 2D visualisation, as well as of effectively highlighting differences in the conformational dynamics directly related to biological functions.

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Lorenzi, Magali. "Etude des transitions structurales dans les protéines flexibles par marquage de spin suivi par spectroscopie de Résonance Paramagnétique Electronique (RPE)." Thesis, Aix-Marseille 1, 2011. http://www.theses.fr/2011AIX10139/document.

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L’étude des transitions structurales dans les protéines est d’un intérêt crucial car ces transformations sont impliquées dans de nombreux processus biologiques essentiels. De tels phénomènes structuraux peuvent être à l’origine de propriétés remarquables dans les protéines flexibles ou désordonnées, propriétés difficilement accessibles par les techniques structurales usuelles. Le marquage de spin couplé à la spectroscopie de résonance paramagnétique électronique (RPE) est une technique bien adaptée pour l’étude de ces transitions structurales. L’insertion d’un radical nitroxyde sur une cystéine, naturelle ou introduite par mutagenèse dirigée, située à un endroit clé de la protéine permet d’obtenir des informations locales sur les changements structuraux éventuels provoqués par l’ajout d’un partenaire.Cette technique a été appliquée à deux systèmes biologiques comportant un degré de flexibilité différent. La flexibilité de la protéine chaperon NarJ, intervenant dans la biogenèse du complexe Nitrate Réductase de la bactérie Escherichia coli, a été étudiée en présence de son peptide partenaire. Ces études ont permis d’une part de déterminer le site d’interaction et d’autre part, de montrer que l’association des deux partenaires entraîne un verrouillage dans une conformation préférentielle de NarJ. Le deuxième sujet d’étude est la protéine CP12 de Chlamydomonas reinhardtii, intervenant dans la régulation d’un complexe supramoléculaire du cycle de Calvin. La CP12 s’apparente à une protéine intrinsèquement désordonnée, ayant la particularité de posséder des cystéines naturelles et fonctionnelles. Le marquage classique a permis de mettre en évidence un nouveau rôle de son partenaire et de montrer que la CP12 garde un caractère désordonné dans le complexe. Par ailleurs, cette protéine a servi de système d’étude pour développer une nouvelle stratégie de marquage sur Tyrosine et démontrer sa faisabilité
The study of structural transitions in proteins is of crucial interest because these transformations are involved in many biological processes. Such structural phenomena can be the source of remarkable properties in flexible or disordered proteins, properties hardly accessible by conventional structural techniques. Site-directed spin labeling combined with electron paramagnetic resonance spectroscopy (EPR) is a technique well suited for the study of these structural transitions. The insertion of a nitroxide reagent on a cysteine, natural or introduced by site-directed mutagenesis, located in a key position of a protein provides local information on possible structural changes induced by the addition of a partner. This technique was applied on two biological systems with a different degree of flexibility. The flexibility of NarJ, a chaperon protein involved in the biogenesis of the complex nitrate reductase of Escherichia coli was studied in the presence of its peptide partner. These studies enabled us to determine the interaction site and to show that the association of the two partners induced a locked conformation of NarJ. The second system is the CP12 protein of Chlamydomonas reinhardtii, involved in the regulation of a supramolecular complex of the Calvin cycle. CP12 shares some similarities with the intrinsically disordered protein but having natural and functional cysteines. The conventional labeling allowed us to highlight a new role of its partner and to demonstrate that CP12 remains disordered in the complex. Moreover, this protein was used as a model system to develop a new labeling strategy on tyrosine and to demonstrate its feasibility

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32

Leis, Simon [Verfasser], Martin [Akademischer Betreuer] Zacharias, and Iris [Akademischer Betreuer] Antes. "Impact of Protein conformational Changes on Molecular Docking - Design of a Docking Approach including Receptor Flexibility / Simon Leis. Gutachter: Iris Antes. Betreuer: Martin Zacharias." München : Universitätsbibliothek der TU München, 2012. http://d-nb.info/1030099464/34.

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D'Cunha, Cassian. "A molecular simulation study of the utility of methyl groups as probes of protein packing and flexibility and maintaining the computational environment for computational biomolecular research." FIU Digital Commons, 2003. http://digitalcommons.fiu.edu/etd/3020.

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Methyl groups are ubiquitous in proteins and may be useful probes of protein packing and flexibility. The purpose of this research is to determine a practical computational method for predicting methyl group dynamics and to determine its correlation with protein packing and flexibility. Molecular dynamics simulations were performed on a set of four crystalline amino acids and peptides (Ala, Leu, Val, and CLA) and the proteins staphylococcal nuclease (SNase) and HIV-1 protease. The dynamics of methyl rotation was quantified and compared with the results of NMR experiments and rotational barrier calculations. This study required considerable computational resources, and hence the setup and maintenance of the computational environment is an important aspect of the research. Installation of hardware and software, customization of software, maintenance of user accounts, and the writing of scripts to optimize use of the computer resources was required.

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Santiago, Daniel Navarrete. "Use and Development of Computational Tools in Drug Discovery: From Small Molecules to Cyclic Peptides." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4398.

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The scope of this work focuses on computationally modeling compounds with protein structures. While the impetus of drug discovery is the innovation of new therapeutic molecules, it also involves distinguishing molecules that would not be an effective drug. This can be achieved by inventing new tools or by refining old tools. Virtual screening (VS, also called docking), the computational modeling of a molecule in a receptor structure, is a staple in predicting a molecule's affinity for an intended target. In our Virtual Target Screening system (also called inverse-docking), VS is used to find high-affinity targets, which can potentially explain absorption, distribution, metabolism, and excretion (ADME) of a molecule of interest in the human body. The next project, low-mode docking (LD), attempts to improve VS by incorporating protein flexibility into traditional docking where a static receptor structure has potential to produce poor results due to incorrectly predicted ligand poses. Finally, VS, performed mostly on small molecules, is scaled up to cyclic peptides by employing Monte Carlo simulations and molecular dynamics to mimic the steps of small molecule VS. The first project discussed is Virtual Target Screening (also called inverse-docking) where a small molecule is virtually screened against a library of protein structures. Predicting receptors to which a synthesized compound may bind would give insights to drug repurposing, metabolism, toxicity, and lead optimization. Our protocol calibrates each protein entry with a diverse set of small molecule structures, the NCI Diversity Set I. Our test set, 20 kinase inhibitors, was predicted to have a high percentage of kinase "hits" among approximately 1500 protein structures. Further, approved drugs within the test set generally had better rates of kinase hits. Next, normal mode analysis (NMA), which can computationally describe the fundamental motions of a receptor structure, is utilized to approach the rigid body bias problem in traditional docking techniques. Traditional docking involves the selection of a static receptor structure for VS; however, protein structures are dynamic. Simulation of the induced fit effect in protein-ligand binding events is modeled by full articulation of the approximated large-scale low-frequency normal modes of vibration, or "low-modes," coupled with the docking of a ligand structure. Low-mode dockings of 40 cyclin dependent 2 (CDK2) inhibitors into 54 low-modes of CDK2 yielded minimum root-mean-square deviation (RMSD) values of 1.82 – 1.20 Å when compared to known coordinate data. The choice of pose is currently limited to docking score, however, with ligand pose RMSD values of 3.87 – 2.07 Å. When compared to corresponding traditional dockings with RMSD values of 5.89 – 2.33 Å, low-mode docking was more accurate. The last discussion involves the rational docking of a cyclic peptide to the murine double minute 2 (MDM2) oncoprotein. The affinity for a cyclic peptide (synthesized by Priyesh Jain, McLaughin Lab, University of South Florida), PJ-8-73, in MDM2 was found to be within an order of magnitude of a cyclic peptide from the Robinson Lab at the University of Zurich in Switzerland. Both are Β-hairpin cyclic peptides with IC50 values of 650 nm and 140 nm, respectively. Using the co-crystalized structure of the Robinson peptide (PDB 2AXI), we modeled the McLaughlin peptide based on an important interaction of the 6-chloro-tryptophan residue of the Robinson peptide occupying the same pocket in MDM2 as the tryptophan residue by the native p53 transactivation helical domain. By preserving this interaction in initial cyclic peptide poses, the resulting pose of PJ-8-73 structure in MDM2 possessed comparable active site residue contacts and surface area. These protocols will aid medical research by using computer technology to reduce cost and time. VTS utilizes a unique structural and statistical calibration to virtually assay thousands of protein structures to predict high affinity binding. Determining unintended protein targets aids in creating more effective drugs. In low-mode docking, the accuracy of virtual screening was increased by including the fundamental motions of proteins. This newfound accuracy can decrease false negative results common in virtual screening. Lastly, docking techniques, usually for small molecules, were applied to larger peptide molecules. These modifications allow for the prediction of peptide therapeutics in protein-protein interaction modulation, a growing interest in medicine. Impactful in their own ways, these procedures contribute to the discovery of drugs, whether they are small molecules or cyclic peptides.

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PASI, MARCO. "A dynamical perspective on cold-adapted enzymes at the molecolar level." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2009. http://hdl.handle.net/10281/7729.

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Some organisms, especially unicellular, have adapted to extreme temperature conditions, and are capable of proliferating in such environments mainly thanks to the optimisation of their enzymatic repertoire. Cold-adapted (psychrophilic) enzymes are invariably characterised by high catalytic activity at low temperatures, necessary to endure the exponential reduction of the speed of chemical reactions in these conditions, and by low thermal stability. Numerous studies aimed at understanding the mechanisms of such adaptation at the molecular level agree that the high turnover observed for psychrophilic enzymes results from a decrease of the activation enthalpy of the catalysed reaction, which in turn is achieved structurally by decreasing the number and strength of the enthalpically stabilised interactions which must be broken to reach the transition state. Since these interactions are also involved in the stabilisation of the enzyme structure, their decrease has the effect of increasing structural flexibility, as reflected by the low thermal stability found for psychrophilic enzymes. Flexibility seems thus to have a fundamental role in enzymatic cold adaptation, and its study by means of Molecular Dynamics (MD) simulations provides a detailed description taking rigorously into account its dynamical character. I report the results of comparative MD studies performed on homologous mesophilic and psychrophilic enzymes, and mutants thereof, to investigate the molecular bases of cold adaptation. Long, multi-replica and explicit-solvent MD simulations have been compared in particular in terms of molecular flexibility and the dynamics of intramolecular interactions. Results show that differences in the dynamic character of the compared enzymes are found in loops surrounding the active site or substrate-binding cleft. In the case of chloride-dependent alpha-amylases, the comparison of the cold-active enzyme from Pseudoalteromonas Haloplanktis with its mesophilic porcine homologue shows that modulation of the size and flexibility of these loops cause the immediate surroundings of the active site to be comparatively more flexible in the psychrophilic enzyme. Detailed analysis of these enzymes active-site dynamics reveals that elements previously identified through X-ray crystallography as involved in substrate binding in both enzymes undergo concerted motions that may be linked to catalysis. The comparison of psychrophilic and mesophilic isoforms of trypsin from Salmo Salar shows that the cold-adapted enzyme presents fewer interdomain interactions and enhanced localised flexibility in regions close to the catalytic site. Notably, these regions fit well with the pattern of protein flexibility previously reported for psychrophilic elastases. These results indicate that specific sites within the serine-protease fold can be considered hot spots of cold-adaptation and that psychrophilic trypsins and elastases have independently discovered similar molecular strategies to optimise flexibility at low temperatures. This evidence of evolutionary convergence underlines the importance of extending intrafamiliar comparative studies to unveil general features of how enzymes adapt their dynamical properties low temperatures. Molecular dynamics studies using all-atomic models, as those presented herein, have proven their effectiveness in this context, but their computational requirement hampers their applicability as the size of the test set increases. For these reasons it would be relevant to devise a simplified ("coarse-grain") approach to perform comparative analyses at a higher throughput than is currently feasible. To evaluate the level of simplification most suitable for this application, a "coarse-grain" approach has been adopted to study the mechanical properties of trypsins, taking into account results obtained using all-atomic models. The results of these comparisons are driving the development of a multi-scale "coarse-grain" approach that combines the required efficiency and accuracy.

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Nguyen, Thi Minh-Ha. "Molecular recognition of ubiquitin and Lysine 63 linked diubiquitin by STAM2 : the effect of the linkers length and flexibility." Thesis, Lyon, 2019. https://n2t.net/ark:/47881/m6668chz.

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Les interactions protéine-proteine sont considérées comme un domaine de recherche important puisqu’elles contrôlent la plupart des processus cellulaires. Chez les cellules eucaryotes, les protéines multi-domaines (MDP), constituées d’au moins deux domaines, représentent plus de 70 % des protéines. Au sein d’une MDP, ces domaines peuvent être identiques ou différents et sont reliés par un segment intrinsèquement désordonné de longueur et de flexibilité variable. Ces protéines peuvent alors adopter de multiples conformations dans l’espace et interagir de manière spécifique avec leurs partenaires biologiques. Malgré de nombreux efforts de recherche dans le domaine, certaines questions restent encore non résolues ou nécessitent une étude approfondie. Mon projet de recherche est d’étudier et de définir le rôle des segments intrinsèquement désordonnés de la protéine STAM2 (Signal transducing adapter molecule 2) impliquée dans la machinerie ESCRT (Endosomal Sorting Complexe Required for Transport) , première étape dans le processus de dégradation lysosomale. Plus précisément, l’étude se focalise sur les effets de la flexibilité et la dynamique de ces segments dans le cas du processus de reconnaissance moléculaire entre STAM2 et l’ubiquitine ou di-ubiquitine. Différents mutants ont alors été conçus : soit avec un domaine totalement ou partiellement supprimé, soit avec un raccourcissement ou une suppression complète du segment ou soit avec de multiples mutations dans la séquence peptidique du segment. Ces différents construits ont été analysés en utilisant une combinaison de techniques biophysiques telles que la relaxation de spin par résonance magnétique nucléaire (RMN), la diffusion des rayons X aux petits angles (SAXS) et le dichroïsme circulaire (CD). Il a alors été démontré qu’une altération du segment désordonné peut entraîner un changement de la dynamique de la protéine et/ou un changement conformationnel. La modification de ce segment influe sur le mouvement inter-domaine et modifie l’affinité entre les construits de STAM2 et la di-ubiquitine sans modifier l’intégrité de chaque domaine et de leur site de liaison. En résumé, les segments intrinsèquement désordonnés procurent une certaine plasticité à la protéine ce qui lui permet de s’adapter et de remplir sa fonction biologique. Il est alors possible d’imaginer dans un futur proche que ces segments soient la nouvelle génération de cibles thérapeutiques pouvant réduire ou supprimer certaines interactions nocives
Protein-protein interaction is considered as an important field of research, as it is the key to control variable cell processes and pathways. In eucaryotic cells, multidomain proteins (MDPs), which consist of more than one domain, take up over 70 % of the pool. Those identical or different domains of a MDP are connected to each other by a linker of variable length and flexibility. For long flexible linker, it allows the protein to sample a wide range of conformation and to adjust interaction in a subtle way. Despite numerous efforts of research on the field, some issues remain unanswered or require further investigation. As part of this thesis, my work aims to define the role taken by the intrinsically disordered linker within MDPs. For that purpose, the STAM2 (Signal transducing adapter molecule 2) protein of the ESCRT (Endosomal Sorting Complexes Required for Transport) machinery was chosen to examine the effect of the flexibility and dynamics of the linker regions on the molecular recognition with ubiquitin and Lysine63-linked di-ubiquitin (K63-Ub2). Such efforts were carried out by designing specific mutants altering the linker regions in different ways. The various truncated versions undergo half or complete deletion of a domain or have their linker either shortened, deleted or modified in the amino acid composition. With a combination of the several biophysical methods namely NMR (Nuclear Magnetic Resonance) spin relaxation, SAXS (Small Angle X-ray Scattering) and CD (Circular Dichroism), the study has demonstrated that the alteration in the linker region modifies the flexibility and the dynamics of the protein, one among them possibly introduces slight change in conformation. Furthermore, the modification of the linker has an impact on the inter-domain motion and alter binding affinities between STAM2 constructs and di-ubiquitin without affecting domains integrity or binding sites. In brief, disordered linkers provide plasticity to the protein, which allow adaptability and specificity to molecular recognition process. As a further application, the linkers included in multidomain proteins could also be the next generation of druggable target as their modification may reduce or completely abolish interactions

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Alonso, Hernan, and hernan alonso@anu edu au. "Computer Modelling and Simulations of Enzymes and their Mechanisms." The Australian National University. The John Curtin School of Medical Research, 2006. http://thesis.anu.edu.au./public/adt-ANU20061212.161155.

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Although the tremendous catalytic power of enzymes is widely recognized, their exact mechanisms of action are still a source of debate. In order to elucidate the origin of their power, it is necessary to look at individual residues and atoms, and establish their contribution to ligand binding, activation, and reaction. Given the present limitations of experimental techniques, only computational tools allow for such detailed analysis. During my PhD studies I have applied a variety of computational methods, reviewed in Chapter 2, to the study of two enzymes: DfrB dihydrofolate reductase (DHFR) and methyltetrahydrofolate: corrinoid/iron-sulfur protein methyltransferase (MeTr). ¶ The DfrB enzyme has intrigued microbiologists since it was discovered thirty years ago, because of its simple structure, enzymatic inefficiency, and its insensitivity to trimethoprim. This bacterial enzyme shows neither structural nor sequence similarity with its chromosomal counterpart, despite both catalysing the reduction of dihydrofolate (DHF) using NADPH as a cofactor. As numerous attempts to obtain experimental structures of an enzyme ternary complex have been unsuccessful, I combined docking studies and molecular dynamics simulations to produce a reliable model of the reactive DfrBDHFNADPH complex. These results, combined with published empirical data, showed that multiple binding modes of the ligands are possible within DfrB. ¶ Comprehensive sequence and structural analysis provided further insight into the DfrB family. The presence of the dfrB genes within integrons and their level of sequence conservation suggest that they are old structures that had been diverging well before the introduction of trimethoprim. Each monomer of the tetrameric active enzyme presents an SH3-fold domain; this is a eukaryotic auxiliary domain never found before as the sole domain of a protein, let alone as the catalytic one. Overall, DfrB DHFR seems to be a poorly adapted catalyst, a minimalistic enzyme that promotes the reaction by facilitating the approach of the ligands rather than by using specific catalytic residues. ¶ MeTr initiates the Wood-Ljungdahl pathway of anaerobic CO2 fixation. It catalyses the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH3THF) to the cobalt centre of a corrinoid/iron-sulfur protein. For the reaction to occur, the N5 position of CH3THF is expected to be activated by protonation. As experimental studies have led to conflicting suggestions, computational approaches were used to address the activation mechanism. ¶ Initially, I tested the accuracy of quantum mechanical (QM) methods to predict protonation positions and pKas of pterin, folate, and their analogues. Then, different protonation states of CH3THF and active-site aspartic residues were analysed. Fragment QM calculations suggested that the pKa of N5 in CH3THF is likely to increase upon protein binding. Further, ONIOM calculations which accounted for the complete protein structure indicated that active-site aspartic residues are likely to be protonated before the ligand. Finally, solvation and binding free energies of several protonated forms of CH3THF were compared using the thermodynamic integration approach. Taken together, these preliminary results suggest that further work with particular emphasis on the protonation state of active-site aspartic residues is needed in order to elucidate the protonation and activation mechanism of CH3THF within MeTr.

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Alves, Ariane Ferreira Nunes. "Um método computacional para estimar afinidades entre proteínas flexíveis e pequenos ligantes." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-08052013-144801/.

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Métodos computacionais são usados para gerar estruturas de complexo proteína-ligante e estimar suas afinidades. Esse trabalho investigou como as diferentes representações da flexibilidade proteica afetam as poses obtidas por ancoragem molecular e as afinidades atribuídas a essas poses. Os mutantes L99A e L99A/M102Q da lisozima T4 foram escolhidos como sistemas modelo. Um descritor para predição de afinidades baseado na aproximação de energia de interação linear (LIE) foi parametrizado especificamente para ligantes da lisozima e foi usado para estimar as afinidades. A proteína foi representada como um grupo de estruturas cristalográficas ou de estruturas de trajetória de dinâmica molecular. O campo de força OPLS-AA para modelar a proteína e os ligantes e a aproximação de Born generalizada para modelar o solvente foram empregados. O descritor de afinidades parametrizado resultou em desvios médios entre afinidades experimentais e calculadas de 1,8 kcal/mol para um conjunto de testes. O descritor teve desempenho satisfatório na separação entre poses cristalográficas e poses falso-positivo e na identificação de poses falso-positivo. Experimentos de agrupamento de complexos realizados com o objetivo de reduzir o custo computacional para estimar afinidades apresentaram resultados insatisfatórios. As melhores aproximações da teoria do ligante implícito propostas aqui para estimar afinidades consideram conjuntos de estruturas de receptor com o mesmo peso. Configurações de ligante também apresentam o mesmo peso ou são dominadas por uma única configuração. A representação da flexibilidade requer um tratamento estatístico adequado para estimativa de afinidades. Aqui, a associação entre LIE e a teoria do ligante implícito mostrou-se frutífera.
Computational methods are used to generate protein-ligand complex structures and estimate their binding affinities. This work investigated how different representations of protein flexibility affect poses obtained by molecular docking and the affinities attributed to these poses. T4 lysozyme mutants L99A and L99A/M102Q were chosen as model systems. A descriptor for prediction of affinities based on linear interaction energy (LIE) approximation was parametrized specifically to lysozyme ligands and was used to estimate affinities. The protein was represented as a group of crystal structures or as structures from a molecular dynamics trajectory. OPLS-AA force field was used to model protein and ligands and the Generalized Born approximation was used to model solvent. The parametrized affinity descriptor resulted in average deviations between experimental and calculated affinities of 1.8 kcal/mol for a test set. Descriptor performance was satisfactory in the separation between crystal poses and false-positive ones and in the identification of false-positive poses. Clustering of complexes was tried out to reduce computational cost to estimate affinities, but results were poor. The best approximations to the implicit ligand theory proposed here in order to estimate affinities consider groups of receptor structures with the same weight. Ligand configurations also have the same weight or are dominated by only one configuration. The representation of protein flexibility requires an adequate statistical treatment when used to estimate affinities. Here, the linking between LIE and the implicit ligand theory proved itself useful.

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Neto, Antonio Marinho da Silva. "Geometria diferencial e teoria da informação aplicada a análise de ensembles conformacionais de proteínas." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-07032018-150722/.

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Um dos maiores desafios atuais na biologia estrutural é como lidar com flexibilidade de proteínas. Além do desafio experimental, uma limitação teórica é a falta de uma linguagem matemática conveniente para representação do espaço conformacional de proteínas. As representações mais populares apresentam diversas limitações, que se refletem nas dificuldades associadas à análise de ensembles conformacionais. Nesse contexto, a aplicação de geometria diferencial (GD) e teoria da informação (TI) foi pouco explorada. Neste trabalho investigamos o uso de descritores de GD e TI como uma representação matemática do espaço conformacional de proteínas aplicada à análise de ensembles conformacionais. O cálculo dos descritores de GD consiste em representar o backbone de proteínas como curvas espaciais e caracterizá-las utilizando os seus valores de curvatura, κ, e torção, τ . Baseado nesses valores, definimos medidas de flexibilidade, de distância entre conformações e aplicamos uma estratégia de clustering para identificação de estados conformacionais. Para permitir a aplicação de TI, desenvolvemos um sistema de codificação desses descritores para expressar cada conformação por uma sequência de símbolos finitos. A partir dessas sequências, definimos uma medida da informação associada a um resíduo, Rres, e a uma conformação, Rconf. Para investigar sua eficácia, aplicamos os métodos propostos aos ensembles conformacionais de três sistemas testes: 1) Ubiquitina, 2) E1-DBD do HPV18 e 3) as etapas de formação do complexo c-Myb-KIX. A análise da representação por geometria diferencial se mostrou igualmente eficaz ou superior aos métodos comumente utilizados em todos os sistemas analisados. O método é especialmente útil para monitoramento de estabilidade de hélices e para análise de proteínas e regiões muito flexíveis, pois evita a necessidade de sobreposição estrutural. Os valores de Rconf se apresentaram úteis para análise de processos de enovelamento e resíduos próximos a regiões funcionais tendem a apresentar maiores valores Rres. No entanto, o papel desses resíduos é incerto e mais estudos são necessários para determinar se há e qual é seu real significado. Apesar disso, as medidas de informação se mostraram úteis para comparação de estados conformacionais e permitem levantar hipóteses testáveis em laboratório. Por fim, a representação por GD é computacionalmente conveniente, intuitiva, evita todas as limitações dos métodos popularmente utilizados e se mostrou eficaz para análise de ensembles conformacionais.
One of the major challenges of modern structural biology is how to deal with protein flexibility. Besides the experimental difficulties, a relatively overlooked theoretical challenge is the lack of a proper mathematical language to represent proteín conformational space. The most popular representations have severe limitations, which reflects on the difficulties associated with conformational ensemble analyses. However, differential geometry (GD) and information theory (TI) can help to overcome such difficulties and were not well explored in this context. Here we investigate the usage of DG and TI as a mathematical representation of protein conformational space applied to the analyses of conformational ensembles. The DG descriptors calculation consists of representing protein backbone as a spatial curve and describes it by its curvature, κ, and torsion, τ . Based on those values, the distance between conformation and flexibility measurements were defined and a clustering algorithm was applied to identify conformational states. For the application of TI, a coding system for DG descriptors was developed to express each conformation as a sequence of finite symbols. Based on those sequences, information measurements associated to a residue, Rres, and to a conformation, Rconf , were defined. To investigate its efficacy, the proposed method was applied to conformation ensembles of three test systems: 1) Ubiquitin, 2) E1-DBD of HPV18 and 3) the steps of c-Myb-KIX binding. The DG analyses show equally good or superior performance when compared with popular methods on all tested system. In addition, the methods are especially useful to monitoring helix stability and analyses of very flexible proteins (or regions), since avoids the necessity of superposing structures. The values of Rconf are useful to compare different steps of a folding process and residues near regions involved in binding events tend to present higher values of Rres. However, those residues importance is uncertain and further studies are necessary to determinate if and how those can contribute to protein function. Nevertheless, the information measurements were informative on the comparison of compare conformational states and allow to formulate a testable hypothesis. On the other hand, the GD representation is computationally convenient, intuitive and avoid most of the limitations of the popular method applied to conformational ensemble analyses.

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Gruza, Jan. "Modélisation de la flexibilité des glycannes complexes en solution et en interaction avec des protéines." Grenoble 1, 1998. http://www.theses.fr/1998GRE10158.

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Le comportement conformationnel des glycannes, et donc leur flexibilite, peuvent influencer directement la reactivite chimique et l'activite biologique de ces molecules. Dans ce travail, la modelisation moleculaire d'une serie de glycannes de complexite croissante a ete realisee en utilisant plusieurs methodes d'exploration de l'espace conformationnel, dont la methode heuristique cicada, associees a differents champs de force : mm3 et amber avec plusieurs parametrisations disponibles pour les glycannes. La validation des modeles a ete apportee par des methodes theoriques de chimie quantique et des methodes experimentales telles que la resonance magnetique nucleaire et la cristallographie aux rayons x. Pour les monosaccharides, les comportements conformationnels de deux cycles furanose ont ete etudies par plusieurs methodes et les resultats ont ete compares aux structures cristallines connues. Les constantes de couplage #3j#h#-#h intracycliques ont ete calculees et comparees aux donnees de rmn. Cette approche a permis de determiner les conformations de plus basse energie ainsi que de demontrer la grande flexibilite de ces systemes. Le comportement conformationnel de la liaison glycosidique a ete etudie par modelisation moleculaire de deux disaccharides et d'un heptasaccharide. Ces etudes ont demontre la capacite de la methode cicada a explorer des espaces conformationnels tres complexes de facon rapide et efficace. Les donnees rmn et cristallographiques ont confirme l'existence des modeles de plus basse energie. Afin d'etudier les interactions proteine-glucide, un modele tridimensionnel d'un complexe entre la lectine de lathyrus ochrus et un nonasaccharide biantenne et flucosyle a ete construit. Les comportements conformationnels du nonasaccharide libre et complexe ont ete etablis en utilisant les methodes conformationnelles validees sur les molecules plus petites. Les resultats de la modelisation ont pu etre compares avec les donnees cristallographiques de plusieurs complexes lectine/oligosaccharides. Les limites des methodes de modelisation moleculaire utilisees dans cette etude sont discutees.

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Illingworth, C. J. R. "Structural and electrostatic flexibility in proteins:-computational approaches to ligand binding." Thesis, University of Essex, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486625.

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A significant proportion of our understanding of protein-ligand interactions comes about through static pictures, generated through X-ray crystallography. However, in vivo, such interactions occur in a dynamic environment, characterised by flexibility in both the physical , structure, and the electrostatic properties of the molecules involved. Here we assess the importance of such flexibility in the process ofligand binding. Firstly, examining flexibility in ligand confonnation, we apply an algorithmic approach to a representative sample of proteases, and demonstrate that proteases selectively bind ligands in an extended confonnation. Secondly, through the development of a measure ofthe spatial flexibility of residues in a protein structure, we produce a means of predicting the location of ligand binding sites, and, appiying this to the structure of rhodopsin, generate a result that is consistent with recent experimental work on the binding of chlorine to a novel binding site. Finally, we t~rn to a substantial investigation ofthe importance of flexibility in the electronic distribution of a molecule, examining this through a method of modelling molecular mechanics polarisation through atom-centred point charges.

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Sekhi, Ikram. "Développement d'un alphabet structural intégrant la flexibilité des structures protéiques." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC084/document.

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L’objectif de cette thèse est de proposer un Alphabet Structural (AS) permettant une caractérisation fine et précise des structures tridimensionnelles (3D) des protéines, à l’aide des chaînes de Markov cachées (HMM) qui permettent de prendre en compte la logique issue de l’enchaînement des fragments structuraux en intégrant l’augmentation des conformations 3D des structures protéiques désormais disponibles dans la banque de données de la Protein Data Bank (PDB). Nous proposons dans cette thèse un nouvel alphabet, améliorant l’alphabet structural HMM-SA27,appelé SAFlex (Structural Alphabet Flexibility), dans le but de prendre en compte l’incertitude des données (données manquantes dans les fichiers PDB) et la redondance des structures protéiques. Le nouvel alphabet structural SAFlex obtenu propose donc un nouveau modèle d’encodage rigoureux et robuste. Cet encodage permet de prendre en compte l’incertitude des données en proposant trois options d’encodages : le Maximum a posteriori (MAP), la distribution marginale a posteriori (POST)et le nombre effectif de lettres à chaque position donnée (NEFF). SAFlex fournit également un encodage consensus à partir de différentes réplications (chaînes multiples, monomères et homomères) d’une même protéine. Il permet ainsi la détection de la variabilité structurale entre celles-ci. Les avancées méthodologiques ainsi que l’obtention de l’alphabet SAFlex constituent les contributions principales de ce travail de thèse. Nous présentons aussi le nouveau parser de la PDB (SAFlex-PDB) et nous démontrons que notre parser a un intérêt aussi bien sur le plan qualitatif (détection de diverses erreurs)que quantitatif (rapidité et parallélisation) en le comparant avec deux autres parsers très connus dans le domaine (Biopython et BioJava). Nous proposons également à la communauté scientifique un site web mettant en ligne ce nouvel alphabet structural SAFlex. Ce site web représente la contribution concrète de cette thèse alors que le parser SAFlex-PDB représente une contribution importante pour le fonctionnement du site web proposé. Cette caractérisation précise des conformations 3D et la prise en compte de la redondance des informations 3D disponibles, fournies par SAFlex, a en effet un impact très important pour la modélisation de la conformation et de la variabilité des structures 3D, des boucles protéiques et des régions d’interface avec différents partenaires, impliqués dans la fonction des protéines
The purpose of this PhD is to provide a Structural Alphabet (SA) for more accurate characterization of protein three-dimensional (3D) structures as well as integrating the increasing protein 3D structure information currently available in the Protein Data Bank (PDB). The SA also takes into consideration the logic behind the structural fragments sequence by using the hidden Markov Model (HMM). In this PhD, we describe a new structural alphabet, improving the existing HMM-SA27 structural alphabet, called SAFlex (Structural Alphabet Flexibility), in order to take into account the uncertainty of data (missing data in PDB files) and the redundancy of protein structures. The new SAFlex structural alphabet obtained therefore offers a new, rigorous and robust encoding model. This encoding takes into account the encoding uncertainty by providing three encoding options: the maximum a posteriori (MAP), the marginal posterior distribution (POST), and the effective number of letters at each given position (NEFF). SAFlex also provides and builds a consensus encoding from different replicates (multiple chains, monomers and several homomers) of a single protein. It thus allows the detection of structural variability between different chains. The methodological advances and the achievement of the SAFlex alphabet are the main contributions of this PhD. We also present the new PDB parser(SAFlex-PDB) and we demonstrate that our parser is therefore interesting both qualitative (detection of various errors) and quantitative terms (program optimization and parallelization) by comparing it with two other parsers well-known in the area of Bioinformatics (Biopython and BioJava). The SAFlex structural alphabet is being made available to the scientific community by providing a website. The SAFlex web server represents the concrete contribution of this PhD while the SAFlex-PDB parser represents an important contribution to the proper function of the proposed website. Here, we describe the functions and the interfaces of the SAFlex web server. The SAFlex can be used in various fashions for a protein tertiary structure of a given PDB format file; it can be used for encoding the 3D structure, identifying and predicting missing data. Hence, it is the only alphabet able to encode and predict the missing data in a 3D protein structure to date. Finally, these improvements; are promising to explore increasing protein redundancy data and obtain useful quantification of their flexibility

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Mejia, Tamayo Verónica. "Propriétés volumétriques des Arabinogalactan-protéines d'exsudats de gommes d'Acacia." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTG060/document.

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La gomme d’Acacia est l’une des plus anciennes gommes naturelles dans le monde et la plus connue. Elle est définie comme l’exsudat gommeux produit par les arbres d’Acacia senegal et Acacia seyal. Les gommes d’Acacia sont composées d’arabinogalactanes protéines (AGPs), faiblement chargés, hyperbranchés avec une forte proportion de sucres (90%) et d’environ 1-3% de protéines et de minéraux. Malgré ses nombreuses applications industrielles, les connaissances sur ses propriétés volumétriques (hydrostatiques et hydrodynamiques) restent à améliorer. Ces propriétés peuvent être liées à la flexibilité et l’hydratation des molécules qui déterminent les propriétés fonctionnelles importantes comme les propriétés interfaciales. L'objectif de cette thèse est l’étude des propriétés volumétriques d’AGPs de la gomme d’Acacia. L’étude a été faite sur les principales variétés des gommes d’Acacia, A. senegal et A. seyal, ainsi que des fractions macromoléculaires d’A. senegal, obtenues par la chromatographie d'interaction hydrophobe et d'échange ionique. Les principaux résultats ont montré que les AGPs de gomme d’Acacia ont une structure semi flexible. De plus, des différences dans la flexibilité et l’hydratation entre les fractions d’AGPs ont été montré. Ces différences ont été expliquées par leurs différences en composition, polarité, masse molaire, forme et conformation. De plus, un comportement intermédiaire entre des protéines et des polysaccharides linéaires ont été montré. Finalement, un effet des agrégats d’AGPs sur les propriétés volumétriques a été mis en avance
Acacia gum is the oldest and most widely known and used gum, it is a dried gummy exudate from the leaves and branches of the Acacia senegal and Acacia seyal trees. Acacia gums are weakly charged, amphiphilic hyperbranched arabinogalactan-proteins (AGPs). They are composed of about 90% polysaccharides and from 1-3% of proteins and minerals. In spite of the widely spread of industrial usage of A. gums, their volumetric properties (hydrostatic and hydrodynamic) have not been well studied. These properties have been linked to important properties such as flexibility and hydration of the molecule, which are related to important functional properties of A. gums (e. g. interfacial properties). The main objective of this PhD thesis was to study the volumetric properties of AGPs from Acacia gums exudates. For this effect, the main commercial species, A. senegal and A. seyal, and the macromolecular fractions of the former, obtained via hydrophobic interaction and ionic exchange chromatographies were studied. The main results showed that AGPs from Acacia gums have a semi-flexible structure. However, differences in their flexibility and hydration were seen among AGP fractions. These differences were explained based on their composition, polarity, molar mass, shape and conformation. Furthermore, an intermediate behavior between proteins and linear polysaccharides was evidenced. In addition, an effect of the presence of AGP based aggregates on the volumetric properties was seen

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Ruzmetov, Talant A. "THE ROLE OF CHAIN FLEXIBILITY AND CONFORMATIONALDYNAMICS ON INTRINSICALLY DISORDERED PROTEINASSOCIATION." Kent State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=kent1564588247414425.

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Kahler, Anna [Verfasser], Heinrich [Akademischer Betreuer] Sticht, Timothy [Gutachter] Clark, and Heinrich [Gutachter] Sticht. "Intrinsic Flexibility and Structural Stability of Proteins / Anna Kahler ; Gutachter: Timothy Clark, Heinrich Sticht ; Betreuer: Heinrich Sticht." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2018. http://d-nb.info/1173422668/34.

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Schütz, Denise [Verfasser], Thomas F. [Gutachter] Prisner, and Volker [Gutachter] Dötsch. "Conformational flexibility of multi-domain proteins determined by pulsed EPR spectroscopy / Denise Schütz ; Gutachter: Thomas F. Prisner, Volker Dötsch." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2017. http://d-nb.info/1148548262/34.

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Menzel, Anja [Verfasser], Milton T. [Akademischer Betreuer] Stubbs, Ulrich [Akademischer Betreuer] Baumann, and Renate [Akademischer Betreuer] Ulbrich-Hofmann. "Flexibilität und Spezifität in Protein-Protein-Wechselwirkungen am Beispiel der Komplexbildung von Trypsin-Varianten und dem Proteaseinhibitor Eglin C / Anja Menzel. Betreuer: Milton T. Stubbs ; Ulrich Baumann ; Renate Ulbrich-Hofmann." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2009. http://d-nb.info/1024937135/34.

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Bauman, Mariia A. "Characterizing the Effect of Conformational Changes in the Protein SufU on its Ability to Enhance Enzymatic Activity of the Cysteine Desulfurase SufS in Streptococcus mutans." Bowling Green State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1467906490.

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Kapoor, Shobhna [Verfasser], Roland [Akademischer Betreuer] Winter, and Martin [Akademischer Betreuer] Engelhard. "Biophysical insights into the Ras-membrane ballet: orientational flexibility, conformational substates and mechanosensitivity of Ras proteins / Shobhna Kapoor. Betreuer: Roland Winter. Gutachter: Martin Engelhard." Dortmund : Universitätsbibliothek Dortmund, 2013. http://d-nb.info/1099297656/34.

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Silva, Júlio César da. "Estudos de macromoléculas biológicas parcialmente desestruturadas usando espalhamento de raios-X." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/277988.

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Orientador: Iris Concepción Linares de Torriani
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin
Made available in DSpace on 2018-08-15T22:30:16Z (GMT). No. of bitstreams: 1 Silva_JulioCesarda_D.pdf: 7791031 bytes, checksum: 5711654f743b7d6fb045861e9239ad8c (MD5) Previous issue date: 2010
Resumo: As técnicas de caracterização estrutural de macromoléculas tradicionais se baseiam no fato de uma macromolécula possuir uma conformação compacta e estruturada. Partes flexíveis ou regiões desordenadas têm sido sempre consideradas como grandes obstáculos para técnicas como a cristalografia de raios-X e a ressonância magnética nuclear (RMN). A necessidade de entender a atividade funcional de proteínas nativamente desenoveladas e de proteínas flexíveis com múltiplos domínios tem adquirido grande importância recentemente, mesmo porque essas proteínas desafiam o paradigma de que uma proteína precisa de uma estrutura bem definida para ser funcional. É bem nesse ponto que a técnica de espalhamento de raios-X a baixos ângulos (SAXS) surge oferecendo ferramentas únicas para realizar estudos de macromoléculas flexíveis ou parcialmente desestruturadas, com aplicações muito bem sucedidas em polímeros, matéria mole e macromoléculas em solução. Neste trabalho de tese decidimos enfrentar o desafio de caracterizar proteínas que não possuem uma estrutura bem definida. A teoria do espalhamento mereceu especial cuidado para se adequar tanto aos métodos experimentais da técnica quanto aos tratamentos matemáticos em cálculos usados para estudar esse tipo de proteínas. Apresentamos aqui o estudo de duas proteínas pertencentes à classe das proteínas nativamente desenoveladas: (1) a proteína FEZ1, que é necessária para o crescimento de axônios; (2) a proteína Ki-1/57, que é encontrada em diversas células com câncer principalmente em tumores do sistema linfático. Estudamos também algumas proteínas com múltiplos domínios conectados por regiões flexíveis e que são: (1) duas chaperonas da classe das HSP40 (proteínas Sis1 e Ydj1) juntamente com construções onde alguns domínios dessas proteínas foram cortados; (2) a proteína ribonucléica heterogênea hnRNP-Q que está envolvida em importantes funções do RNA. Experiências de SAXS foram realizadas, fornecendo parâmetros dimensionais e informações de forma dessas proteínas em solução. Modelos de baixa resolução das possíveis conformações foram calculados a partir das curvas de SAXS usando métodos de modelagem ab initio combinados com modelagem de corpos rígidos. Os resultados forneceram informações importantes para elucidar as funções biológicas dessas proteínas. É importante ressaltar que, para realizar os estudos com proteínas em solução, é necessário contar com uma instrumentação adequada e devidamente montada para a aplicação da técnica de SAXS. Para isso, durante o período de desenvolvimento deste doutorado houve um grande investimento na montagem, teste e caracterização de instrumentos, junto à equipe de profissionais do Laboratório Nacional de Luz Síncrotron (LNLS), completando o comissionamento da estação experimental SAXS2 do LNLS
Abstract: The traditional techniques for structural characterization of macromolecules are based on a compact and structured conformation of the macromolecule. Flexible or disordered regions have usually been regarded as a great hindrance to techniques like X-ray protein crystallography and nuclear magnetic resonance (NMR). The need to study functional activity of natively unfolded proteins and flexible multidomain proteins came to the light rather recently, defying the classical structure¿function paradigm where a protein must have a well-defined 3-D structure to be functional. In this type of situation, the small-angle X-ray scattering (SAXS) technique appears as a unique tool to deal with this problem. Indeed, the application of SAXS methods to the characterization of soft matter (e.g. polymers) and macromolecules in solution has already succeeded during the last years. In this work we decided to face the challenge of characterizing proteins that do not have a well defined structure. The SAXS experimental technique as well as the mathematical methods and calculations needed special attention in order to be correctly applied to study the specific problem of unstructured proteins in solution. Thus, it was possible to find evidence of the structural details of these proteins and obtain a low resolution 3-D average structure. Here we present the study of two proteins that belong to the group of natively unfolded proteins: (1) The FEZ1 protein, which is necessary for axon growth, and (2) the proteins indentified as Ki-1/57, which is found in diverse cancer cells mainly in lymphatic systems tumors. We also studied some flexible multidomain proteins: (1) two chaperones from the groups of HSP40 (the proteínas Sis1 e Ydj1), and two mutant constructions where some domains were deleted; (2) the heterogeneous ribonucleoprotein hnRNP-Q which is related to an array of important functions of RNA. Several SAXS experiments were performed providing overall parameters and important shape information about those proteins in solution. Low resolution models for the possible conformations of these proteins were restored from the SAXS curves using ab initio modeling methods combined with rigid body modeling. The SAXS results provided a unique structural background for the biologists to deal with the function of these proteins. SAXS experiments with proteins in solution demand the use of a specific instrumentation properly developed for those studies. So, it is important to mention that, throughout the duration of this doctorate, specific instrumentation development and testing was done together with the technical staff of the Brazilian Synchrotron Light Laboratory (LNLS, Campinas, SP, Brazil), collaborating with the commissioning of the new SAXS2 workstation, completed in 2008
Doutorado
Física
Doutor em Ciências

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Dissertations / Theses on the topic 'Protein flexibility'

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