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Park, Changmoon Goddard William A. "Protein design and simulation Part I. Protein design. Part II. Protein simulation /." Diss., Pasadena, Calif. : California Institute of Technology, 1993. http://resolver.caltech.edu/CaltechTHESIS:11112009-114142428.
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Thesis (Ph. D.)--California Institute of Technology, 1993. UM #93-25,374.Advisor names found in the Acknowledgements pages of the thesis. Title from home page. Viewed 01/15/2010. Includes bibliographical references.
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Kwan, Ann H. Y. "Protein design based on a PHD scaffold." Connect to full text, 2004. http://setis.library.usyd.edu.au/adt/public_html/adt-NU/public/adt-NU20041202.102526/index.html.
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Thesis (Ph. D.)--School of Molecular and Microbial Biosciences, Faculty of Science, University of Sydney, 2004.Chapter headings on separately inserted unnumbered cream coloured leaves. Bibliography: leaves 122-135.
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Baas, Tracey Lynn. "The design, synthesis, and characterization of template assembled synthetic proteins /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/11561.
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Barua, Bipasha. "Design and study of Trp-cage miniproteins /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/8533.
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Dantas, Gautam. "In silico protein evolution by intelligent design : creating new and improved protein structures /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/9236.
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Badger, David B. "Design and Synthesis of Protein-Protein Interaction Inhibitor Scaffolds." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/3964.
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Many currently relevant diseases such as cancer arise from altered biological pathways that rely on protein-protein interactions. The proteins involved in these interactions contain certain functional domains that are responsible for the protein's biological activities. These domains consist of secondary structural elements such as α-helices and Β-sheets which are at the heart of the protein's biological activity. Therefore, designing drugs that inhibit protein-protein interactions by binding to these key secondary structural elements should provide an effective treatment for many diseases. Presented in this dissertation are the designs, syntheses, and biological evaluations for both novel α-helix and novel Β-sheet mimics.
The α-helix mimics were designed to inhibit the interactions between the tumor suppressor protein p53 and its inhibitor protein, MDM2. We also targeted the interactions between the Bak/Bcl-xL proteins. Using the knowledge gained from Hamilton's 1,4-terphenylene scaffold, we designed our inhibitors to be non-peptidic small molecule α-helix mimics. These molecules were designed to bind to the NH2-terminal domain of MDM2 protein thus preventing it from binding to the p53 protein thereby allowing p53 to induce apoptosis. The α-helix mimetic scaffold is designed around a central functionalized pyridazine ring while maintaining the appropriate distances between the ith, ith+4, and ith+7 positions of a natural alpha helix.
The Β-sheet mimics were designed as inhibitors for the integrin mediated extracellular matrix cell adhesion found in Multiple Myeloma. We have designed, synthesized, and incorporated novel Β-turns to induce the formation of Β-hairpins as well as to cyclize the peptides in order to increase their binding affinities and reduce proteolytic cleavage. Given that many protein-protein interactions occur through hydrophobic interactions; our primary Β-turn promoter was designed with the ability to alter the Β-hairpin's hydrophobicity depending on the sulfonyl group used in the turn. The synthesis of several different sulfonyl chlorides for use in our Β-turn promoter is included in this section. We have also provided a detailed structural analysis and characterization of these new cyclic peptides via NMR and CD spectrometry. Using standard 2D NMR methods, we have elucidated the 3D conformation of several peptides in solution. We have also studied the structure activity relationships (SAR) for these cyclic peptides and then correlated these results with those obtained from the NMR studies.
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Datta, Deepshikha Goddard William A. "Protein-ligand interactions : docking, design and protein conformational change /." Diss., Pasadena, Calif. : California Institute of Technology, 2003. http://resolver.caltech.edu/CaltechETD:etd-03242003-111426.
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Bazzoli, A. "Protein structure prediction and protein design with evolutionary algorithms." Doctoral thesis, Università degli Studi di Milano, 2009. http://hdl.handle.net/2434/64478.
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Campbell, Sean Thomas. "Protein Engineering for Biochemical Interrogation and System Design." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/560940.
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Proteins are intimately involved in almost every cellular phenomenon, from life to death. Understanding the interactions of proteins with each other and other macromolecules and the ability to rationally redesign them to improve their activities or control their function are of considerable current interest. Split-protein methodologies provide an avenue for achieving many of these goals. Since the original discovery of conditionally activated split-ubiquitin, the field has grown exponentially to include the activities of over a dozen different proteins. The flexibility of the systems has resulted in their use across a wide spectrum, both literally and figuratively, to primarily screen, visualize and quantitate macromolecular interactions in a variety of biological systems. In another arena, there is significant interest the apoptosis-regulating proteins: the Bcl-2 family. These proteins are found in many cell types and control, through expression levels as well as other mechanisms, the apoptotic state of a protein as governed by intrinsic death signals generated from such sources as DNA damage and viral infection. The apoptotic function of these proteins are mainly governed by a single type of interaction: the helix:receptor binding of the BH3-Only helices to the anti-apoptotic receptor proteins. While this often promiscuous helix:receptor interaction has received much scrutiny, the nature of the anti-apoptotic binding pocket, especially with regard to the specific residues that govern the interaction, has been lacking. With the high sensitivity and rapid analysis platform afforded by the cell-free split-luciferase analysis methodology, we devised and carried out the first systematic and large scale alanine mutagenesis of all five major anti-apoptotic members of the Bcl-2 family, validated these results both with biophysical methods as well as correlation with previous studies. Our results help explain how different receptors can bind a wide range of helices and also uncovered details regarding binding that are not possible with structural or computational analysis alone. In a second area of research, we have utilized the interaction of BH3 helices and their receptors for designing small molecule controlled protein kinases and phosphatases. In this protein design area, BH3-Only helices were inserted using a knowledge based approach into particular loops within both a protein kinase and a protein phosphatase. The BH3-Only helix interaction with added receptors, such as Bcl-xL provided an allosteric switch for turning-off the activity of the helix-inserted enzymes. The activity of the enzymes could then be turned-on by the addition of a cell-permeable small molecule that is known to bind the receptor. This plug-and-play design was demonstrated to be successful for two very different enzyme classes and likely provides a general and tunable biological element for controlling the activity of one or more proteins and enzymes in a biochemical networks.
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Rege, Nischay Kiran. "THE UN-DESIGN AND DESIGN OF INSULIN: STRUCTURAL EVOLUTIONWITH APPLICATION TO THERAPEUTIC DESIGN." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1531429783955495.
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Traore, Seydou. "Computational approaches toward protein design." Thesis, Toulouse, INSA, 2014. http://www.theses.fr/2014ISAT0033/document.
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Le Design computationnel de protéines, en anglais « Computational Protein Design » (CPD), est un champ derecherche récent qui vise à fournir des outils de prédiction pour compléter l'ingénierie des protéines. En effet,outre la compréhension théorique des propriétés physico-chimiques fondamentales et fonctionnelles desprotéines, l’ingénierie des protéines a d’importantes applications dans un large éventail de domaines, y comprisdans la biomédecine, la biotechnologie, la nanobiotechnologie et la conception de composés respectueux del’environnement. Le CPD cherche ainsi à accélérer le design de protéines dotées des propriétés désirées enpermettant le traitement d’espaces de séquences de large taille tout en limitant les coûts financier et humain auniveau expérimental.Pour atteindre cet objectif, le CPD requière trois ingrédients conçus de manière appropriée: 1) une modélisationréaliste du système à remodeler; 2) une définition précise des fonctions objectives permettant de caractériser lafonction biochimique ou la propriété physico-chimique cible; 3) et enfin des méthodes d'optimisation efficacespour gérer de grandes tailles de combinatoire.Dans cette thèse, nous avons abordé le CPD avec une attention particulière portée sur l’optimisationcombinatoire. Dans une première série d'études, nous avons appliqué pour la première fois les méthodesd'optimisation de réseaux de fonctions de coût à la résolution de problèmes de CPD. Nous avons constaté qu’encomparaison des autres méthodes existantes, nos approches apportent une accélération du temps de calcul parplusieurs ordres de grandeur sur un large éventail de cas réels de CPD comprenant le design de la stabilité deprotéines ainsi que de complexes protéine-protéine et protéine-ligand. Un critère pour définir l'espace demutations des résidus a également été introduit afin de biaiser les séquences vers celles attendues par uneévolution naturelle en prenant en compte des propriétés structurales des acides aminés. Les méthodesdéveloppées ont été intégrées dans un logiciel dédié au CPD afin de les rendre plus facilement accessibles à lacommunauté scientifiqueComputational Protein Design (CPD) is a very young research field which aims at providing predictive tools to complementprotein engineering. Indeed, in addition to the theoretical understanding of fundamental properties and function of proteins,protein engineering has important applications in a broad range of fields, including biomedical applications, biotechnology,nanobiotechnology and the design of green reagents. CPD seeks at accelerating the design of proteins with wanted propertiesby enabling the exploration of larger sequence space while limiting the financial and human costs at experimental level.To succeed this endeavor, CPD requires three ingredients to be appropriately conceived: 1) a realistic modeling of the designsystem; 2) an accurate definition of objective functions for the target biochemical function or physico-chemical property; 3)and finally an efficient optimization framework to handle large combinatorial sizes.In this thesis, we addressed CPD problems with a special focus on combinatorial optimization. In a first series of studies, weapplied for the first time the Cost Function Network optimization framework to solve CPD problems and found that incomparison to other existing methods, it brings several orders of magnitude speedup on a wide range of real CPD instancesthat include the stability design of proteins, protein-protein and protein-ligand complexes. A tailored criterion to define themutation space of residues was also introduced in order to constrain output sequences to those expected by natural evolutionthrough the integration of some structural properties of amino acids in the protein environment. The developed methods werefinally integrated into a CPD-dedicated software in order to facilitate its accessibility to the scientific community
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Stafford, Ryan Leonard Grubbs Robert H. "Design of protein-DNA dimerizers /." Diss., Pasadena, Calif. : Caltech, 2008. http://resolver.caltech.edu/CaltechETD:etd-08232007-154048.
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MARCHETTI, FILIPPO. "COMPUTATIONAL STUDIES OF PROTEIN-PROTEIN AND PROTEIN-ANTIBODY INTERACTIONS: IMPLICATION FOR MOLECULAR DESIGN." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/825462.
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High performance computing has opened the possibility to investigate complex systems by simulating their dynamics and study of equilibrium and non-equilibrium settings in realistic settings. Molecular Dynamics (MD) simulations have emerged as one of the privileged methods to disentangle the intricacies of biochemical systems but, despite the validity of Moore’s Law, the timescale of the events that can be simulated has an upper limit of the millisecond with tailor-made computers which is not enough to study some biologically relevant phenomena.
Starting from these considerations, in this thesis, I have set out to develop and validate novel methods to predict the location of potentially interacting surfaces on proteins and to predict the impact of small molecules on the activation vs. the inhibition of proteins’ functional dynamic states. To this end, I have combined physico-chemical approaches to the study of protein dynamics and generate novel approaches that may overcome the current limitations of pure brute force MD simulations.
In the first part of the thesis, I studied methods for the prediction of the residues involved in protein-protein interactions. I presented two different scores, one based on evolutionary information and one based on the energetics of the protein, on a dataset of crystal structures. Both scores have the capability to discriminate the interface region from the rest of the protein in a relevant fraction of cases. Moreover, a comparison of the scores efficacy on distinct protein classes highlights the importance of considering the biological function of the protein on the performance of the method used for the prediction of interface residues. In addition, the energetic method for interface residues prediction is used for the detection of antigenic epitopes on the spike protein of SARS-CoV-2. The regions predicted were confirmed against experimental complexes expanding our understanding of the molecular basis for interactions. In perspective, the acquired knowledge could be used for the design of novel vaccine candidates and diagnostic tools and to increase our readiness in the case of future epidemics. In the second part there, I focussed on the study of two allosteric systems. Firstly a method is presented for the integration of an ensemble docking protocol with a learning classifier for allosteric ligands of the protein Hsp90. The method reaches a good accuracy in classifying the activity of these ligands and this approach seems to reduce the dependency on the chemical similarity of the compounds used for the training. The method is tested on a limited dataset and further developments could be achieved in the future if the library of compounds is increased. In the end, I presented the initial analysis of an allosteric signal for integrinαvβ6 in complex with a pro-TGFβpeptide, with the use of molecular dynamics simulations. The data suggest that the presence of the peptide induces an increased rigidity of the legs of the structure, in particular for a specific domain.
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Long, Stephen. "Combinatorial methods in drug design : towards modulating protein-protein interactions./." St. Lucia, Qld, 2003. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17525.pdf.
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Fuller, Jonathan Christopher. "Computational approaches for drug design at the protein-protein interface." Thesis, University of Leeds, 2010. http://etheses.whiterose.ac.uk/1699/.
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The ability to design drugs that disrupt formation of protein-protein interfaces is of particular interest to the pharmaceutical industry due to its promise for opening an entire new range of drug targets, many of which have already been well characterised in terms of their disease causing effect on the human body. Furthermore these interactions can be involved in many processes unique and essential to bacteria and viruses. We show that pockets on protein-protein interface are smaller but more numerous than those of marketed drugs using a pocket fnding algorithm (Q-SiteFinder). We investigate the similarities and differences between several candidate compounds designed to bind and disrupt protein-protein interfaces and compare to those of current marketed drugs designed to bind more traditional protein targets. We ask the further question as to whether it is possible to better identify pockets on a protein surface as likely to be drug binding. We conclude that it is possible to carefully use random forest machine learning techniques to marginally improve these predictions. However, it is extremely diffcult to use simple physical parameters to provide added information as to the maximal affnity that a small-molecule might be able to achieve in a given binding pocket. Further to the above questions we then investigate the hDM2-p53 system which when disrupted can induce apoptosis in many forms of cancer, making it a target of considerable interest to the pharmaceutical industry. Molecular docking is exploited in order to generate likely structural conformations of oligoamide hDM2-p53 inhibitors which can be used as a starting point for molecular dynamics simulations. These simulations using the AMBER/GAFF force feld are then further developed to perform replica-exchange alchemical free energy calculations using the Bennett Acceptance Ratio non-biased estimator. These simulations are in general shown to be very accurate and show promise in generating hypotheses for novel high-affnity oligoamide compounds.
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Grässlin, Anja. "Protein epitope mimetics as inhibitors of protein-protein interactions and in synthetic vaccine design /." Zürich, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000254199.
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Wood, Christopher Robin Wells. "Computational design of parameterisable protein folds." Thesis, University of Bristol, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.715832.
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Lacroix, Emmanuel. "Protein design: a computer-based approach." Doctoral thesis, Universite Libre de Bruxelles, 1999. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211882.
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Scott, Richard Kennedy. "Protein modelling and associated drug design." Thesis, University of Newcastle Upon Tyne, 1993. http://hdl.handle.net/10443/523.
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Part one covers an investigation into the secondary and tertiary structure of the protein Xylanase found in Pseudomonasfluorescens subspecies cellulosa. Part two documents the Computer Aided Design of Novel Quinazoline Antifolates for the enzyme Thyrnidylate Synthase. Partone Mature Xylanase protein consists of two distinct regions - a cellulose binding domain and a catalytic region, A and B respectively. Computer modelling of tertiary structure from primary sequence and secondary turn information proved difficult in the absence of experimental X-ray crystal data. Consequently,a series of modified proteins bbased on the Xylanase were prepared by Recombinant DNA technology for extraction and purification. The modified proteins were to be used as a bench mark for quantitative and definitive calculation and detennination of the secondary structure of the xylanase. This was to provide an excellent reference point for theoretical modelling of tertiary structure. Part one of the Thesis documents Computer Modelling work and protein purification and extraction of the xylanase. Part two Thymidylate Synthase (TS) exists as dimer with a single active site in each subunit. It has been crystallised in two forms; a "reduced" (major), and "oxidised" (minor) form. The major form of TS contains dUMP covalently bound to cysteine in both active sites and in the presence of CB3717. One active site of the minor form contains dUMP non-covalently bound and in the presence of CB3717 while the other active site contains only inorganic phosphate and CB3717. The active site of TS is a large cavity that binds CB3717 into two possible confirmations. One is seen in the major form and one in the minor form. Part two of my research documents an investigation into enzyme/inhibitor interaction in TS and covers the Computer Aided Design of a series of inhibitors based on the knowledge of the TS active site. Several of these compounds have been put forward as target compounds for synthesis.
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Borghei, Golnaz. "Design of a BRET fluorescent protein." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607666.
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Zollars, Eric Stafford Pierce Niles A. "Force field development in protein design /." Diss., Pasadena, Calif. : Caltech, 2006. http://resolver.caltech.edu/CaltechETD:etd-06052006-155305.
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Rossi, Andrea. "Statistical Mechanics Approach to Protein Design." Doctoral thesis, SISSA, 2000. http://hdl.handle.net/20.500.11767/4329.
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The plan of this thesis is the following. In the first chapter we introduce the most important and basic concept related to protein folding and design. In the second chapter, after introducing some standard lattice models of proteins and heteropolymers, the most important methods of protein design present in the literature are described. In the third chapter we will introduce a novel iterative procedure for protein design and it will be applied to lattice protein models [58]. A different approach based on geometrical criterion [49] will also be presented. In the fourth chapter, we will implement an approximated approach in order to design real protein structures [59]. In this case, it has been possible to compare our designed sequences with real sequences, whose native states are known. The good correlation between natural sequences and designed sequences indicates that the method is very promising.
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Zhang, Guangtao. "Design, synthesis, and evaluation of cholera toxin inhibitors and [alpha]-helix mimetics of dormancy survival regulator /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8485.
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Grigoryan, Gevorg Ph D. Massachusetts Institute of Technology. "Computational approaches for the design and prediction of protein-protein interactions." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38997.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.Includes bibliographical references (leaves 167-187).
There is a large class of applications in computational structural biology for which atomic-level representation is crucial for understanding the underlying biological phenomena, yet explicit atomic-level modeling is computationally prohibitive. Computational protein design, homology modeling, protein interaction prediction, docking and structure recognition are among these applications. Models that are commonly applied to these problems combine atomic-level representation with assumptions and approximations that make them computationally feasible. In this thesis I focus on several aspects of this type of modeling, analyze its limitations, propose improvements and explore applications to the design and prediction of protein-protein interactions.
by Gevorg Grigoryan.
Ph.D.
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Jones, Lisa Michelle. "Using Protein Design to Understand the Role of Electrostatic Interactions on Calcium Binding Affinity and Molecular Recognition." Digital Archive @ GSU, 2008. http://digitalarchive.gsu.edu/chemistry_diss/16.
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Calcium regulates many biological processes through interaction with proteins with different conformational, dynamic, and metal binding properties. Previous studies have shown that the electrostatic environment plays a key role in calcium binding affinity. In this research, we aim to dissect the contribution of the electrostatic environment to calcium binding affinity using protein design. Many natural calcium binding proteins undergo large conformational changes upon calcium binding which hampers the study of these proteins. In addition, cooperativity between multiple calcium binding sites makes it difficult to study site-specific binding affinity. The design of a single calcium binding site into a host system eliminates the difficulties that occur in the study of calcium binding affinity. Using a computer algorithm we have rationally designed several calcium binding sites with a pentagonal bipyramidal geometry in the non-calcium dependent cell adhesion protein CD2 (CD2-D1) to better investigate the key factors that affect calcium binding affinity. The first generation proteins are all in varying electrostatic environments. The conformational and metal binding properties of each of these designed proteins were analyzed. The second generation designed protein, CD2.6D79, was designed based on criteria learned from the first generation proteins. This protein contains a novel calcium binding site with ligands all from the â-strands of the non-calcium dependent cell adhesion protein CD2. The resulting protein maintains native secondary and tertiary packing and folding properties. In addition to its selectivity for calcium over other mono and divalent metal ions, it displays strong metal binding affinities for calcium and its analogues terbium and lanthanum. Furthermore, our designed protein binds CD48, the ligand binding partner of CD2, with an affinity three-fold stronger than CD2. The electrostatic potential of the calcium binding site was modified through mutation to facilitate the study of the effect of electrostatic interactions on calcium binding affinity. Several charge distribution mutants display varying metal binding affinities based on their charge, distance to the calcium binding site, and protein stability. This study will provide insight into the key site factors that control calcium binding affinity and calcium dependent biological function.
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Davey, James A. "Multistate Computational Protein Design: Theories, Methods, and Applications." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/35541.
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Traditional computational protein design (CPD) calculations model sequence perturbations and evaluate their stabilities using a single fixed protein backbone template in an approach referred to as single‐state design (SSD). However, certain design objectives require the explicit consideration of multiple conformational states. Cases where a multistate framework may be advantageous over the single‐state approach include the computer aided discovery of new enzyme substrates, the prediction of protein stabilities, and the design of protein dynamics. These design objectives can be tackled using multistate design (MSD). However, it is often the case
that a design objective requires the consideration of a protein state having no available structure information. For such circumstances the multistate framework cannot be applied. In this thesis I present the development of two template and ensemble preparation methodologies and their application to three projects. The purpose of which is to demonstrate the necessary ensemble modeling strategies to overcome limitations in available structure information. Particular emphasis is placed on the ability to recapitulate experimental data to guide modelling of the design space. Specifically, the use of MSD allowed for the accurate prediction of a methyltransferase recognition motif and new substrates, the prediction of mutant sequence stabilities with quantitative accuracy, and the design of dynamics into the rigid Gβ1 scaffold producing a set of dynamic variants whose tryptophan residue exchanges between two conformations on the millisecond timescale. Implementation of both the ensemble, coordinate perturbation followed by energy minimization (PertMin), and template, rotamer optimization followed by energy minimization (ROM), generation protocols developed here allow for exploration and manipulation of the structure space enabling the success of these applications.
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Sarisky, Catherine Ann Roberts Richard W. "Exploration of the determinants of protein structure and stability by protein design /." Diss., Pasadena, Calif. : California Institute of Technology, 2005. http://resolver.caltech.edu/CaltechETD:etd-05272005-121337.
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McCord, Jennifer Phipps. "Protein Engineering for Biomedicine and Beyond." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/90787.
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Many applications in biomedicine, research, and industry require recognition agents with specificity and selectivity for their target. Protein engineering enables the design of scaffolds that can bind targets of interest while increasing their stability, and expanding the scope of applications in which these scaffolds will be useful.
Repeat proteins are instrumental in a wide variety of biological processes, including the recognition of pathogen-associated molecular patterns by the immune system. A number of successes using alternative immune system repeat protein scaffolds have expanded the scope of recognition agents available for targeting glycans and glycoproteins in particular. We have analyzed the innate immune genes of a freshwater polyp and found that they contained particularly long contiguous domains with high sequence similarity between repeats in these domains. We undertook statistical design to create a binding protein based on the H. magnipapillata innate immune TPR proteins.
My second research project focused on creating a protein to bind cellulose, as it is the most abundant and inexpensive source of biomass and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept lignocellulosic fuels from competing commercially with starch-based biofuels. In recent years a strategy to protect processing enzymes with synergistic proteins emerged to reduce the amount of enzyme necessary for lignocellulosic biofuel production. Simultaneously, protein engineering approaches have been developed to optimize proteins for function and stability enabling the use of proteins under non-native conditions and the unique conditions required for any necessary application. We designed a consensus protein based on the carbohydrate-binding protein domain CBM1 that will bind to cellulosic materials. The resulting designed protein is a stable monomeric protein that binds to both microcrystalline cellulose and amorphous regenerated cellulose thin films. By studying small changes to the binding site, we can better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications such as enhancing the saccharification of lignocellulosic feedstock for biofuel production.
Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal scaffolds for tissue engineering and wound healing. However, the extraction process leads to protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Recombinant keratin biomaterials are free from these disadvantages, while heterologous expression of these proteins allows us to manipulate the primary sequence. We endeavored to add an RGD sequence to facilitate cell adhesion to the recombinant keratin proteins, to demonstrate an example of useful sequence modification.Doctor of Philosophy
Many applications in medicine and research require molecular sensors that bind their target tightly and selectively, even in complex mixtures. Mammalian antibodies are the best-studied examples of these sensors, but problems with the stability, expense, and selectivity of these antibodies have led to the development of alternatives. In the search for better sensors, repeat proteins have emerged as one promising class, as repeat proteins are relatively simple to design while being able to bind specifically and selectively to their targets. However, a drawback of commonly used designed repeat proteins is that their targets are typically restricted to proteins, while many targets of biomedical interest are sugars, such as those that are responsible for blood types. Repeat proteins from the immune system, on the other hand, bind targets of many different types. We looked at the unusual immune system of a freshwater polyp as inspiration to design a new repeat protein to recognize nonprotein targets. My second research project focused on binding cellulose, as it is the most abundant and inexpensive source of biological matter and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept cellulose-based fuels from competing commercially with biofuel made from corn and other starchy plants. One strategy to lower costs relies on using helper proteins to reduce the amount of enzyme needed to break down the cellulose, as enzymes are the most expensive part of processing. We designed such a protein for this function to be more stable than natural proteins currently used. The resulting designed protein binds to multiple cellulose structures. Designing a protein from scratch also allows us to study small changes to the binding site, allowing us to better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal for tissue engineering and wound healing applications. However, the process by which these proteins are extracted from hair leads to some protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Making these proteins synthetically allows us to have pure starting material, and lets us add new features to the proteins, which translates into materials better tailored for their applications. We discuss here one example, in which we added a cell-binding motif to a keratin protein sequence.
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Denarie, Laurent. "Robotics-inspired methods to enhance protein design." Phd thesis, Toulouse, INPT, 2017. http://oatao.univ-toulouse.fr/18677/1/Denarie.pdf.
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The ability to design proteins with specific properties would yield great progress in pharmacology and bio-technologies. Methods to design proteins have been developed since a few decades and some relevant achievements have been made including de novo protein design. Yet, current approaches suffer some serious limitations. By not taking protein’s backbone motions into account, they fail at capturing some of the properties of the candidate design and cannot guarantee that the solution will in fact be stable for the goal conformation. Besides, although multi-states design methods have been proposed, they do not guarantee that a feasible trajectory between those states exists, which means that design problem involving state transitions are out of reach of the current methods. This thesis investigates how robotics-inspired algorithms can be used to efficiently explore the conformational landscape of a protein aiming to enhance protein design methods by introducing additional backbone flexibility. This work also provides first milestones towards protein motion design.
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Hong, Eun-Jong 1975. "Exact rotamer optimization for computational protein design." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/44421.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.Includes bibliographical references (leaves 235-244).
The search for the global minimum energy conformation (GMEC) of protein side chains is an important computational challenge in protein structure prediction and design. Using rotamer models, the problem is formulated as a NP-hard optimization problem. Dead-end elimination (DEE) methods combined with systematic A* search (DEE/A*) have proven useful, but may not be strong enough as we attempt to solve protein design problems where a large number of similar rotamers is eligible and the network of interactions between residues is dense. In this thesis, we present an exact solution method, named BroMAP (branch-and-bound rotamer optimization using MAP estimation), for such protein design problems. The design goal of BroMAP is to be able to expand smaller search trees than conventional branch-and-bound methods while performing only a moderate amount of computation in each node, thereby reducing the total running time. To achieve that, BroMAP attempts reduction of the problem size within each node through DEE and elimination by energy lower bounds from approximate maximurn-a-posteriori (MAP) estimation. The lower bounds are also exploited in branching and subproblem selection for fast discovery of strong upper bounds. Our computational results show that BroMAP tends to be faster than DEE/A* for large protein design cases. BroMAP also solved cases that were not solvable by DEE/A* within the maximum allowed time, and did not incur significant disadvantage for cases where DEE/A* performed well. In the second part of the thesis, we explore several ways of improving the energy lower bounds by using Lagrangian relaxation. Through computational experiments, solving the dual problem derived from cyclic subgraphs, such as triplets, is shown to produce stronger lower bounds than using the tree-reweighted max-product algorithm.
(cont.) In the second approach, the Lagrangian relaxation is tightened through addition of violated valid inequalities. Finally, we suggest a way of computing individual lower bounds using the dual method. The preliminary results from evaluating BroMAP employing the dual bounds suggest that the use of the strengthened bounds does not in general improve the running time of BroMAP due to the longer running time of the dual method.
by Eun-Jong Hong.
Ph.D.
31
Biddle, Jason Charles. "Methods and applications in computational protein design." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/61792.
Full textAbstract:
Thesis (S.M.)--Massachusetts Institute of Technology, Computation for Design and Optimization Program, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (p. 107-111).
In this thesis, we summarize our work on applications and methods for computational protein design. First, we apply computational protein design to address the problem of degradation in stored proteins. Specifically, we target cysteine, asparagine, glutamine, and methionine amino acid residues to reduce or eliminate a protein's susceptibility to degradation via aggregation, deamidation, and oxidation. We demonstrate this technique on a subset of degradation-prone amino acids in phosphotriesterase, an enzyme that hydrolyzes toxic organophosphates including pesticides and chemical warfare agents. Second, we introduce BroMAP/A*, an exhaustive branch-and- bound search technique with enumeration. We compare performance of BroMAP/A* to DEE/A*, the current standard for conformational search with enumeration in the protein design community. When limited computational resources are available, DEE/A* sometimes fails to find the global minimum energy conformation and/or enumerate the lowest-energy conformations for large designs. Given the same computational resources, we show how BroMAP/A* is able to solve large designs by efficiently dividing the search space into small, solvable subproblems.
by Jason Charles Biddle.
S.M.
32
Boas, F. Edward. "Physics-based design of protein-ligand binding /." May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
Full text
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Kwan, Ann Hau Yu. "Protein Design Based on a PHD Scaffold." Thesis, The University of Sydney, 2004. http://hdl.handle.net/2123/564.
Full textAbstract:
The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society.
34
Kwan, Ann Hau Yu. "Protein Design Based on a PHD Scaffold." University of Sydney. Molecular and Microbial Biosciences, 2004. http://hdl.handle.net/2123/564.
Full textAbstract:
The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society.
35
Hu, Yaogang. "Design and Synthesis of Bioactive Peptidomimetics." Scholar Commons, 2015. https://scholarcommons.usf.edu/etd/5504.
Full textAbstract:
Protein-Protein Interactions (PPIs) play a very important role in biological functions and therefore the inhibition of specific Protein-Protein Interactions has a huge therapeutic value. The most successful small molecular PPIs inhibitors do not fit with the prevalent `Rule of Five' drug profile. To overcome the disadvantages of small molecular PPIs inhibitors, peptide based PPIs inhibitors were developed. Herein we describe the development of a new class of peptidomimetics AA-peptides. The AApeptides were designed based on chiral PNA backbone. Substitution of nucleobases yields AApeptides that are resistant to proteolysis and capable of mimicking peptides. Two types of AApeptides were discussed in this dissertation "α-AApeptides" and "γ-AApeptides". The AApeptides were shown to disrupt p53/MDM2 protein-protein interaction and tomimic fMLF tripeptide to target G protein-coupled formyl peptide receptors (FPRs). Moreover, the lipidated α-AApeptides can mimic the structure and function of natural antimicrobial lipopeptides and show broad-spectrum activity against both Gram-positive and Gram-negative bacteria. Lastly I have designed and synthesized a serials of phosphopeptides to disrupt cancer related STAT3-STAT3 dimerization.
36
Phan, Jamie. "Investigating protein folding by the de novo design of an α-helix oligomer." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/859.
Full textAbstract:
Proteins are composed of a unique sequence of amino acids, whose order guides a protein to adopt its particular fold and perform a specific function. It has been shown that a protein's 3-dimensional structure is embedded within its primary sequence. The problem that remains elusive to biochemists is how a protein's primary sequence directs the folding to adopt such a specific conformation. In an attempt to gain a better understanding of protein folding, my research tests a novel model of protein packing using protein design. The model defines the knob-socket construct as the fundamental unit of packing within protein structure. The knob-socket model characterizes packing specificity in terms of amino acid preferences for sockets in different environments: sockets filled with a knob are involved in inter-helical interactions and free sockets are involved in intra-helical interactions. Equipped with this knowledge, I sought to design a unique protein, Ksα1.1, completely de novo. The sequence was selected to induce helix formation with a predefined tertiary packing interface. Circular dichroism showed that Ksα1.1 formed α-helical secondary structure as intended. The nuclear magnetic resonance studies demonstrated formation of a high order oligomer with increased protein concentration. These results and analysis prove that the knob-socket model is a predictive model for all α-helical protein packing. More importantly, the knob-socket model introduces a new protein design method that can potentially hold a solution to the folding problem.
37
Park, Chihyo. "Combinatorial design and synthesis of peptidomimics and small molecules for protein-protein interactions." Texas A&M University, 2006. http://hdl.handle.net/1969.1/4692.
Full textAbstract:
The solid phase combinatorial method is an excellent tool for the modulation of
protein-protein interactions through focused library generations. Nucleophilic aromatic
substitution reactions with an iodinated template on solid phase has opened a door for
easy and pure libraries of 13-22 membered medium and macrocyclic peptidomimetics.
These peptide mimics showed promising activities for tyrosine kinase receptors.
Iodine functionality can then be used to modify the products, on the resin, via
Sonogashira and Suzuki couplings and presumably through other organometallic
catalysis. The coupled products can have conformational biases that differ from the
iodinated macrocycles. These coupling reactions also provide a means to introduce
additional pharmacophores and to adjust the solubilities of the products.
The fluorinated template also gave libraries of cyclic peptidomimetics on solid phase
in good yields and purities. These libraries have improved water solubility over the
iodinated libraries. The 3-fluorinated template yielded better results than the 5-
fluorinated template. Some compounds showed biological activities in cell survival
assays providing strong support of our approach to mimic external ò-turn sequences in
target proteins.
Intrasite dimerization with 1,5-hexadiyne gave a homodimer as a byproduct. Solidphase
synthesis of bivalent turn mimics with fluorescent tags has been demonstrated.
The key feature of this synthetic route is that homo- and hetero-dimers can be formed
chemoselectively from unprotected monomeric precursors. The dimerization reaction is
very mild and versatile, as only potassium carbonate is required to affect the coupling.
Solution phase library synthesis of small molecule mimics is presented. Some
monomers of full sequence mimics have been prepared to afford dimer generations. Theses monomers were combined with linker handles to afford diverse length of dimers.
Final combination of monomers to make bivalent compounds is in progress.
38
Chen, Tsan-Chou Scott. "Design of protein-protein interaction specificity using computational methods and experimental library screening." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/70386.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Cataloged from PDF version of thesis.
Includes bibliographical references.
Computational design of protein-protein interaction specificity is a powerful tool to examine and expand our understanding about how protein sequence determines interaction specificity. It also has many applications in basic bioscience and biotechnology. One of the major challenges for design is that current scoring functions relying on general physical principles do not always make reliable predictions about interaction specificity. In this thesis I described application of two approaches to address this problem. The first approach sought to improve scoring functions with experimental interaction specificity data related to the protein family of design interest. I used this approach to design inhibitor peptides against the viral bZIP protein BZLF 1. Specificity against design self-interaction was considered in the study. The second approach exploited the power of experimental library screening to characterize a large number of designed sequences at once, increasing the overall probability of identifying successful designs. I presented a novel framework for such library design approach and applied it to the design of anti-apoptotic Bcl-2 proteins with novel interaction specificity toward BH3 peptides. Finally I proposed how these two approaches can be combined together to further enhance our design capabilities.
by Tsan-Chou Scott Chen.
Ph.D.
39
Watkins, Andrew M. "An in silico pipeline for the design of peptidomimetic protein-protein interaction inhibitors." Thesis, New York University, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10188557.
Full textAbstract:
Protein-protein interactions have historically been branded “undruggable” due to their intrinsic challenges above and beyond protein-small molecule interactions. Incrementally, system after system has been approached by a variety of specialized design strategies. Still, the vast majority of interactions are intractable, and the profusion of individualized strategies leave few general approaches that might be able to extend to recalcitrant systems.
The ecosystem of tools available for developing inhibitors of protein-protein interactions suggests a potential modular strategy for proceeding from protein structure to plausible interaction inhibitors. My dissertation describes an analysis of all the protein-protein interactions containing key interfacial structural motifs found in protein structures catalogued by the Protein Data Bank. This work provides both data on extant protein interactions and specific conclusions regarding directions for further peptidomimetic design. We describe the incorporation of our lab’s peptidomimetic scaffolds into Rosetta and the validation of those methods against valuable biological systems. Finally, I chronicle substantial extension to Rosetta’s capacity to accurately model and design peptidomimetic structures.
40
Rufino, Stephen Duarte. "Analysis, comparison and prediction of protein structure." Thesis, Birkbeck (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243648.
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Durani, Venuka. "The Cycle of Protein Engineering: Bioinformatics Design of Two Dimeric Proteins and Computational Design of a Small Globular Domain." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338311626.
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Kouvatsos, Nikolaos. "Characterisation of rabbit ileal lipid binding protein and design of new β-scaffold proteins." Thesis, University of Nottingham, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442293.
Full text
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Larsson, Andreas. "Antiadhesive agents targeting uropathogenic Escherichia coli : Multivariate studies of protein-protein and protein-carbohydrate interactions." Doctoral thesis, Umeå : Dept. of Chemistry, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-314.
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44
Legault, Sandrine. "Investigating Different Rational Design Approaches to Increase Brightness in Red Fluorescent Proteins." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42740.
Full textAbstract:
Red fluorescent proteins (RFPs) are used extensively in biological research because their longer emission wavelengths are less phototoxic and allow deeper imaging of animal tissue. However, far-red RFPs generally display low brightness, emphasizing the need to develop brighter variants. Here, we investigate three approaches to rigidify the RFP chromophore to increase the quantum yield, and thereby brightness. We first used computational protein design on a maturation-efficient mRojo-VHSV variant previously engineered in our lab to introduce a Superdecker motif, a parallel pi-stack comprising aromatic residue side chains and the phenolate moiety of the chromophore, which we hypothesized would enhance chromophore packing and reduce non-radiative decay. The best mutants identified showed up to 1.7-fold higher quantum yield at pH 9, relative to their parent protein. We next postulated that brightness could be further increased by rigidifying the chromophore via branched aliphatic residues. Computational protein design was performed on a dim mCherry variant, mRojoA, followed by directed evolution on the brightest mutant. The combination of these methodologies yielded mSandy2, the brightest Discosoma-derived monomeric RFP with an emission maximum above 600 nm. Finally, we aimed to increase brightness by focusing on positions where residue rigidity correlated to quantum yield in mCherry-related RFPs according to NMR data that had been previously acquired in our lab. Combinatorial site-saturation mutagenesis was performed on two different surface patches of mCherry at positions 144/145/198 and 194/196/220. Our results demonstrated that surface residues may not be adequate targets for this approach. Altogether, the work herein presents unique rational design methodologies that can be used to increase brightness in RFPs.
45
Phan, Jamie. "Investigating protein folding by the de novo design of an α-helix oligomer : a thesis." Scholarly Commons, 2001. https://scholarlycommons.pacific.edu/uop_etds/859.
Full textAbstract:
Proteins are composed of a unique sequence of amino acids, whose order guides a protein to adopt its particular fold and perform a specific function. It has been shown that a protein's 3-dimensional structure is embedded within its primary sequence. The problem that remains elusive to biochemists is how a protein's primary sequence directs the folding to adopt such a specific conformation. In an attempt to gain a better understanding of protein folding, my research tests a novel model of protein packing using protein design. The model defines the knob-socket construct as the fundamental unit of packing within protein structure. The knob-socket model characterizes packing specificity in terms of amino acid preferences for sockets in different environments: sockets filled with a knob are involved in inter-helical interactions and free sockets are involved in intra-helical interactions. Equipped with this knowledge, I sought to design a unique protein, Ksα1.1, completely de novo. The sequence was selected to induce helix formation with a predefined tertiary packing interface. Circular dichroism showed that Ksα1.1 formed α-helical secondary structure as intended. The nuclear magnetic resonance studies demonstrated formation of a high order oligomer with increased protein concentration. These results and analysis prove that the knob-socket model is a predictive model for all α-helical protein packing. More importantly, the knob-socket model introduces a new protein design method that can potentially hold a solution to the folding problem.
46
Blackler, Alissa N. "Design of bone morphogenetic protein 2/nodal chimeras." Diss., [La Jolla] : University of California, San Diego, 2010. http://wwwlib.umi.com/cr/fullcit?p1477885.
Full textAbstract:
Thesis (M. S.)--University of California, San Diego, 2010.Title from first page of PDF file (viewed July 12, 2010). Available via ProQuest Digital Dissertations. Includes bibliographical references (leaves 34-36).
47
Gräslund, Torbjörn. "Protein engineering by directed evolution and rational design /." Stockholm : Tekniska högsk, 2001. http://media.lib.kth.se:8080/kthdisseng.html.
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48
Hong, Wei. "Design and synthesis of protein arginine methyltransferase inhibitors." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/12835/.
Full textAbstract:
Biological methylation is defined as the transfer of a methyl group from S-adenosyl-L-methionine(SAM) to one of a wide range of potential acceptors such as DNA, RNA, protein, hormones and neurotransmitters. Protein arginine methylation is a common post-translational modification facilitated by protein arginine methyltransferases(e.g. PRMTI). The roles of these enzymes in vivo are currently poorly understood. The focus of the project is design and synthesis of PRMT inhibitors with the ultimate goal of evaluating their activities in cells. Preliminary work toward the synthesis of S-adenosyl-trifluoromethyl-L-homocystein and adenosyl 5'-[2-(tert-butoxycarbonylamino)ethyl-trifluoro methyl] thiophenium is described. The ternary crystal structure of PRMTI in complex with S-adenoSyl-L-homocystein(eSAH) and an arginine containing peptide (PBD IOR8) was used to design a series of potential bisubstrate inhibitors of PRMTI. The prototypical SAM analogues bearing guanidine group were sought to replace the reactive sulfonium centre with nitrogen. Analogue synthesis proceeded via successive reductive arnination of Y-arnino-Y-deoxyadenosine and deprotection in good overall yields. An alkyne SAM analogue, 5'-[(S-3-amino-3-carboxypropyl)-propargylaminol-5'-deoxyadenosine was prepared, which underwent efficient Cu(1) catalysed Huisgen reaction to yield a triazole derived SAM analogue 5'-[(S-3-amino-3-carboxypropyl)-[I-(2-guanidinoethyl)-IH-1,2,3-triazol-4-yl]methyl-amino]-5'-deoxyadenosine. Preliminary biological evaluation of the compounds by collaborators Professor Steve Ward and Dr Richard Parry at the University of Bath, confirmed that 5'-[(S-3-amino-3-carboxypropyl)- 3-guanidinopropyl-amino]-5'-deoxyadenosine and 5-[(S-3-amino-3-carboxypropyl)-5-guanidinopentyl-amino]-5'-deoxyadenosine are potent inhibitors of PRMTI but not the lysine methyltransferase SET7. A related N-6 modified SAM analogue 5'-[(S-3-amino-3-carboxypropyl)-3-guanidinopropylamino]-5'-deoxy-N6-(lI-azido-3,6,9-trioxaundecane)-amino adenosine bearing an azide tether was developed with the aim of allowing facile introduction of biotin or fluorescent dyes, using either Staudinger ligation, or Cu(1) catalysed Huisgen reaction to provide compounds that can be used for affinity purification of the target protein or study of its localisation in cells respectively. Finally, progress toward a novel, rapid and enantioselective synthesis of the natural product (+)-sinefungin is reported. Key dihydropyridazine intermediates were generated from adenosyl 5'-propaldehyde, commercially available azodicarboxylate derivatives and ester substituted vinyltriphenylphosphonium salt by successful extension of methodology first reported by Ley and co-workers. Deprotection and ring opening of clihydropyridazine compounds was attempted, and unfortunately we were not able to generate (+)-sinefungin, although it is hoped that this route can be developed to achieve this in the future.
49
Park, Daniel J. (Daniel John) 1979. "Computational tools for including specificity in protein design." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/87286.
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50
Li, Zhong Qi. "Protein secondary structure mimetics : design, synthesis and evaluation." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/38780.
Full text