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Kang, Hyun-Seo, and Michael Sattler. "Capturing dynamic conformational shifts in protein–ligand recognition using integrative structural biology in solution." Emerging Topics in Life Sciences 2, no. 1 (April 20, 2018): 107–19. http://dx.doi.org/10.1042/etls20170090.
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In recent years, a dynamic view of the structure and function of biological macromolecules is emerging, highlighting an essential role of dynamic conformational equilibria to understand molecular mechanisms of biological functions. The structure of a biomolecule, i.e. protein or nucleic acid in solution, is often best described as a dynamic ensemble of conformations, rather than a single structural state. Strikingly, the molecular interactions and functions of the biological macromolecule can then involve a shift between conformations that pre-exist in such an ensemble. Upon external cues, such population shifts of pre-existing conformations allow gradually relaying the signal to the downstream biological events. An inherent feature of this principle is conformational dynamics, where intrinsically disordered regions often play important roles to modulate the conformational ensemble. Unequivocally, solution-state NMR spectroscopy is a powerful technique to study the structure and dynamics of such biomolecules in solution. NMR is increasingly combined with complementary techniques, including fluorescence spectroscopy and small angle scattering. The combination of these techniques provides complementary information about the conformation and dynamics in solution and thus affords a comprehensive description of biomolecular functions and regulations. Here, we illustrate how an integrated approach combining complementary techniques can assess the structure and dynamics of proteins and protein complexes in solution.
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Chakraborty, Arnab, Fabien Deligey, Jenny Quach, Frederic Mentink-Vigier, Ping Wang, and Tuo Wang. "Biomolecular complex viewed by dynamic nuclear polarization solid-state NMR spectroscopy." Biochemical Society Transactions 48, no. 3 (May 7, 2020): 1089–99. http://dx.doi.org/10.1042/bst20191084.
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Solid-state nuclear magnetic resonance (ssNMR) is an indispensable tool for elucidating the structure and dynamics of insoluble and non-crystalline biomolecules. The recent advances in the sensitivity-enhancing technique magic-angle spinning dynamic nuclear polarization (MAS-DNP) have substantially expanded the territory of ssNMR investigations and enabled the detection of polymer interfaces in a cellular environment. This article highlights the emerging MAS-DNP approaches and their applications to the analysis of biomolecular composites and intact cells to determine the folding pathway and ligand binding of proteins, the structural polymorphism of low-populated biopolymers, as well as the physical interactions between carbohydrates, proteins, and lignin. These structural features provide an atomic-level understanding of many cellular processes, promoting the development of better biomaterials and inhibitors. It is anticipated that the capabilities of MAS-DNP in biomolecular and biomaterial research will be further enlarged by the rapid development of instrumentation and methodology.
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Muniyappan, Srinivasan, Yuxi Lin, Young-Ho Lee, and Jin Hae Kim. "17O NMR Spectroscopy: A Novel Probe for Characterizing Protein Structure and Folding." Biology 10, no. 6 (May 21, 2021): 453. http://dx.doi.org/10.3390/biology10060453.
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Oxygen is a key atom that maintains biomolecular structures, regulates various physiological processes, and mediates various biomolecular interactions. Oxygen-17 (17O), therefore, has been proposed as a useful probe that can provide detailed information about various physicochemical features of proteins. This is attributed to the facts that (1) 17O is an active isotope for nuclear magnetic resonance (NMR) spectroscopic approaches; (2) NMR spectroscopy is one of the most suitable tools for characterizing the structural and dynamical features of biomolecules under native-like conditions; and (3) oxygen atoms are frequently involved in essential hydrogen bonds for the structural and functional integrity of proteins or related biomolecules. Although 17O NMR spectroscopic investigations of biomolecules have been considerably hampered due to low natural abundance and the quadruple characteristics of the 17O nucleus, recent theoretical and technical developments have revolutionized this methodology to be optimally poised as a unique and widely applicable tool for determining protein structure and dynamics. In this review, we recapitulate recent developments in 17O NMR spectroscopy to characterize protein structure and folding. In addition, we discuss the highly promising advantages of this methodology over other techniques and explain why further technical and experimental advancements are highly desired.
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Selenko, Philipp. "Quo Vadis Biomolecular NMR Spectroscopy?" International Journal of Molecular Sciences 20, no. 6 (March 14, 2019): 1278. http://dx.doi.org/10.3390/ijms20061278.
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In-cell nuclear magnetic resonance (NMR) spectroscopy offers the possibility to study proteins and other biomolecules at atomic resolution directly in cells. As such, it provides compelling means to complement existing tools in cellular structural biology. Given the dominance of electron microscopy (EM)-based methods in current structure determination routines, I share my personal view about the role of biomolecular NMR spectroscopy in the aftermath of the revolution in resolution. Specifically, I focus on spin-off applications that in-cell NMR has helped to develop and how they may provide broader and more generally applicable routes for future NMR investigations. I discuss the use of ‘static’ and time-resolved solution NMR spectroscopy to detect post-translational protein modifications (PTMs) and to investigate structural consequences that occur in their response. I argue that available examples vindicate the need for collective and systematic efforts to determine post-translationally modified protein structures in the future. Furthermore, I explain my reasoning behind a Quinary Structure Assessment (QSA) initiative to interrogate cellular effects on protein dynamics and transient interactions present in physiological environments.
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van der Wel, Patrick C. A. "New applications of solid-state NMR in structural biology." Emerging Topics in Life Sciences 2, no. 1 (February 23, 2018): 57–67. http://dx.doi.org/10.1042/etls20170088.
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Various recent developments in solid-state nuclear magnetic resonance (ssNMR) spectroscopy have enabled an array of new insights regarding the structure, dynamics, and interactions of biomolecules. In the ever more integrated world of structural biology, ssNMR studies provide structural and dynamic information that is complementary to the data accessible by other means. ssNMR enables the study of samples lacking a crystalline lattice, featuring static as well as dynamic disorder, and does so independent of higher-order symmetry. The present study surveys recent applications of biomolecular ssNMR and examines how this technique is increasingly integrated with other structural biology techniques, such as (cryo) electron microscopy, solution-state NMR, and X-ray crystallography. Traditional ssNMR targets include lipid bilayer membranes and membrane proteins in a lipid bilayer environment. Another classic application has been in the area of protein misfolding and aggregation disorders, where ssNMR has provided essential structural data on oligomers and amyloid fibril aggregates. More recently, the application of ssNMR has expanded to a growing array of biological assemblies, ranging from non-amyloid protein aggregates, protein–protein complexes, viral capsids, and many others. Across these areas, multidimensional magic angle spinning (MAS) ssNMR has, in the last decade, revealed three-dimensional structures, including many that had been inaccessible by other structural biology techniques. Equally important insights in structural and molecular biology derive from the ability of MAS ssNMR to probe information beyond comprehensive protein structures, such as dynamics, solvent exposure, protein–protein interfaces, and substrate–enzyme interactions.
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Jarvis, J. A., I. Haies, M. Lelli, A. J. Rossini, I. Kuprov, M. Carravetta, and P. T. F. Williamson. "Measurement of 14N quadrupole couplings in biomolecular solids using indirect-detection 14N solid-state NMR with DNP." Chemical Communications 53, no. 89 (2017): 12116–19. http://dx.doi.org/10.1039/c7cc03462h.
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Boyd, Patricia S., Janae B. Brown, Joshua D. Brown, Jonathan Catazaro, Issac Chaudry, Pengfei Ding, Xinmei Dong, et al. "NMR Studies of Retroviral Genome Packaging." Viruses 12, no. 10 (September 30, 2020): 1115. http://dx.doi.org/10.3390/v12101115.
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Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.
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Blackledge, M. "Anisotropic Interactions in Solution State NMR : Applications to Biomolecular Structure and Dynamics." EPJ Web of Conferences 30 (2012): 02001. http://dx.doi.org/10.1051/epjconf/20123002001.
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Kim, Tae Hun, Brandon J. Payliss, Michael L. Nosella, Ian T. W. Lee, Yuki Toyama, Julie D. Forman-Kay, and Lewis E. Kay. "Interaction hot spots for phase separation revealed by NMR studies of a CAPRIN1 condensed phase." Proceedings of the National Academy of Sciences 118, no. 23 (June 1, 2021): e2104897118. http://dx.doi.org/10.1073/pnas.2104897118.
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The role of biomolecular condensates in regulating biological function and the importance of dynamic interactions involving intrinsically disordered protein regions (IDRs) in their assembly are increasingly appreciated. While computational and theoretical approaches have provided significant insights into IDR phase behavior, establishing the critical interactions that govern condensation with atomic resolution through experiment is more difficult, given the lack of applicability of standard structural biological tools to study these highly dynamic large-scale associated states. NMR can be a valuable method, but the dynamic and viscous nature of condensed IDRs presents challenges. Using the C-terminal IDR (607 to 709) of CAPRIN1, an RNA-binding protein found in stress granules, P bodies, and messenger RNA transport granules, we have developed and applied a variety of NMR methods for studies of condensed IDR states to provide insights into interactions driving and modulating phase separation. We identify ATP interactions with CAPRIN1 that can enhance or reduce phase separation. We also quantify specific side-chain and backbone interactions within condensed CAPRIN1 that define critical sequences for phase separation and that are reduced by O-GlcNAcylation known to occur during cell cycle and stress. This expanded NMR toolkit that has been developed for characterizing IDR condensates has generated detailed interaction information relevant for understanding CAPRIN1 biology and informing general models of phase separation, with significant potential future applications to illuminate dynamic structure–function relationships in other biological condensates.
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Möbius, Klaus, Wolfgang Lubitz, Nicholas Cox, and Anton Savitsky. "Biomolecular EPR Meets NMR at High Magnetic Fields." Magnetochemistry 4, no. 4 (November 6, 2018): 50. http://dx.doi.org/10.3390/magnetochemistry4040050.
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In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.
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Prestegard, J. H., H. M. Al-Hashimi, and J. R. Tolman. "NMR structures of biomolecules using field oriented media and residual dipolar couplings." Quarterly Reviews of Biophysics 33, no. 4 (November 2000): 371–424. http://dx.doi.org/10.1017/s0033583500003656.
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1. Introduction 3721.1 Residual dipolar couplings as a route to structure and dynamics 3721.2 A brief history of oriented phase high resolution NMR 3742. Theoretical treatment of dipolar interactions 3762.1 Anisotropic interactions as probes of macromolecular structure and dynamics 3762.1.1 The dipolar interaction 3762.1.2 Averaging in the solution state 3772.2 Ordering of a rigid body 3772.2.1 The Saupe order tensor 3782.2.2 Orientational probability distribution function 3802.2.3 The generalized degree of order 3802.3 Molecular structure and internal dynamics 3813. Inducing molecular order in high resolution NMR 3833.1 Tensorial interactions between the magnetic field and anisotropic magnetic susceptibilities 3833.2 Dilute liquid crystal media: a tunable source of order 3843.2.1 Bicelles : from membrane mimics to aligning media 3853.2.2 Filamentous phage 3873.2.3 Transfer of alignment from ordered media to macromolecules 3883.3 Magnetic field alignment 3893.3.1 Paramagnetic assisted alignment 3893.3.2 Advantages of using magnetic alignment 3894. The measurement of residual dipolar couplings 3914.1 Introduction 3914.2 Frequency based methods 3924.2.1 Coupling enhanced pulse schemes 3924.2.2 In phase anti-phase methods (IPAP): 1DNH couplings in proteins 3934.2.3 Exclusive correlated spectroscopy (E-COSY): 1DNH, 1DNC′ and 2DHNC′ 3954.2.4 Extraction of splitting values from the frequency domain 3964.3 Intensity based experiments 3974.3.1 J-Modulated experiments: the measurement of 1DCαHα in proteins 3974.3.2 Phase modulated methods 3994.3.3 Constant time COSY – the measurement of DHH couplings 3994.3.4 Systematic errors in intensity based experiments 4005. Interpretation of residual dipolar coupling data 4015.1 Structure determination protocols utilizing orientational constraints 4015.1.1 The simulated annealing approach 4015.1.2 Order matrix analysis of dipolar couplings 4025.1.3 A discussion of the two approaches 4025.2 Reducing orientational degeneracy 4035.2.1 Multiple alignment media in the simulated annealing approach 4045.2.2 Multiple alignment media in the order matrix approach 4055.3 Simplifying effects arising due to molecular symmetry 4065.4 Database approaches for determining protein structure 4076. Applications to the characterization of macromolecular systems 4086.1 Protein structure refinement 4086.2 Protein domain orientation 4096.3 Oligosaccharides 4136.4 Biomolecular complexes 4156.5 Exchanging systems 4167. Acknowledgements 4188. References 419Within its relatively short history, nuclear magnetic resonance (NMR) spectroscopy has managed to play an important role in the characterization of biomolecular structure. However, the methods on which most of this characterization has been based, Nuclear Overhauser Effect (NOE) measurements for short-range distance constraints and scalar couplings measurements for torsional constraints, have limitations (Wüthrich, 1986). For extended structures, such as DNA helices, for example, propagation of errors in the short distance constraints derived from NOEs leaves the relative orientation of remote parts of the structures poorly defined. Also, the low density of observable protons in contact regions of molecules held together by factors other than hydrophobic packing, leads to poorly defined structures. This is especially true in carbohydrate containing complexes where hydrogen bonds often mediate contacts, and in multi-domain proteins where the area involved in domain–domain contact can also be small. Moreover, most NMR based structural applications are concerned with the characterization of a single, rigid conformer for the final structure. This can leave out important mechanistic information that depends on dynamic aspects and, when motion is present, this can lead to incorrect structural representations. This review focuses on one approach to alleviating some of the existing limitations in NMR based structure determination: the use of constraints derived from the measurement of residual dipolar couplings (D).
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Schlick, Tamar, Rosana Collepardo-Guevara, Leif Arthur Halvorsen, Segun Jung, and Xia Xiao. "Biomolecular modeling and simulation: a field coming of age." Quarterly Reviews of Biophysics 44, no. 2 (January 12, 2011): 191–228. http://dx.doi.org/10.1017/s0033583510000284.
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AbstractWe assess the progress in biomolecular modeling and simulation, focusing on structure prediction and dynamics, by presenting the field's history, metrics for its rise in popularity, early expressed expectations, and current significant applications. The increases in computational power combined with improvements in algorithms and force fields have led to considerable success, especially in protein folding, specificity of ligand/biomolecule interactions, and interpretation of complex experimental phenomena (e.g. NMR relaxation, protein-folding kinetics and multiple conformational states) through the generation of structural hypotheses and pathway mechanisms. Although far from a general automated tool, structure prediction is notable for proteins and RNA that preceded the experiment, especially by knowledge-based approaches. Thus, despite early unrealistic expectations and the realization that computer technology alone will not quickly bridge the gap between experimental and theoretical time frames, ongoing improvements to enhance the accuracy and scope of modeling and simulation are propelling the field onto a productive trajectory to become full partner with experiment and a field on its own right.
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BUCK, MATTHIAS. "Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins." Quarterly Reviews of Biophysics 31, no. 3 (August 1998): 297–355. http://dx.doi.org/10.1017/s003358359800345x.
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Alcohol based cosolvents, such as trifluoroethanol (TFE) have been used for many decades to denature proteins and to stabilize structures in peptides. Nuclear magnetic resonance spectroscopy and site directed mutagenesis have recently made it possible to characterize the effects of TFE and of other alcohols on polypeptide structure and dynamics at high resolution. This review examines such studies, particularly of hen lysozyme and β-lactoglobulin. It presents an overview of what has been learnt about conformational preferences of the polypeptide chain, the interactions that stabilize structures and the nature of the denatured states. The effect of TFE on transition states and on the pathways of protein folding and unfolding are also reviewed. Despite considerable progress there is as yet no single mechanism that accounts for all of the effects TFE and related cosolvents have on polypeptide conformation. However, a number of critical questions are beginning to be answered. Studies with alcohols such as TFE, and ‘cosolvent engineering’ in general, have become valuable tools for probing biomolecular structure, function and dynamics.1. COSOLVENTS: OLD HAT? 2982. HOW DOES TFE WORK? 2992.1 Effect on hydrogen bonding 3002.2 Effect on non-polar sidechains 3012.3 Effect on solvent structure 3023. EFFECTS OF TFE ON (UN-)FOLDING TRANSITIONS 3033.1 Pretransition 3033.2 Transition 3043.3 Posttransition 3053.4 Far UV CD spectroscopic detection of structure 3063.5 Effect with temperature 3063.6 Effect with additional denaturants 3064. THERMODYNAMIC PARAMETERS FROM STRUCTURAL TRANSITIONS OF PEPTIDES AND PROTEINS IN TFE 3075. ADVANCES IN NMR SPECTROSCOPY 3105.1 Chemical shifts 3105.2 3[Jscr ]HNHαcoupling constants 3115.3 Amide hydrogen exchange 3125.4 Nuclear Overhauser Effects (NOEs) 3126. α-HELIX – EVERYWHERE? 3136.1 Intrinsic helix propensity equals helix content? 3136.2 A helix propensity scale for the amino acids in TFE 3146.3 Capping motifs and stop signals 3156.4 Limits and population of helices as seen by CD and NMR 3167. TURNS 3178. β-HAIRPINS AND SHEETS 3179. ‘CLUSTERS’ OF SIDECHAINS 32010. THE TFE DENATURED STATE OF β-LACTOGLOBULIN 32111. THE TFE DENATURED STATE OF HEN LYSOZYME 32412. TERTIARY STRUCTURE, DISULPHIDES, DYNAMICS AND COMPACTNESS 32713. PROSPECTS FOR STRUCTURE CALCULATION 32814. EFFECT OF TFE ON QUATERNARY STRUCTURE 32915. EFFECT ON TFE ON UN- AND REFOLDING KINETICS 33016. OTHER USES 33616.1 Mimicking membranes and protein receptors 33616.2 Solubilizing peptides and proteins 33616.3 Cosolvents as helpers for protein folding? 33816.4 Modifying protein dynamics and catalysis 33816.5 Effects on nucleic acids 33916.6 Effects on lipid bilayers and micelles 33916.7 Future applications 33917. CONCLUSIONS: TFE – WHAT IS IT GOOD FOR? 34018. ACKNOWLEDGMENTS 34019. REFERENCES 340
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Härd, Torleif. "NMR studies of protein–nucleic acid complexes: structures, solvation, dynamics and coupled protein folding." Quarterly Reviews of Biophysics 32, no. 1 (February 1999): 57–98. http://dx.doi.org/10.1017/s0033583599003509.
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Many basic events which concern management and manipulation of genes within a cell – for instance transcription, replication and recombination – rely on specific interactions between proteins and nucleic acids. Such interactions are also essential for many house-keeping functions, like packing and unpacking of DNA in chromatin and assembly of ribosomes. Moreover, the details of protein–nucleic acid interplay is essential for understanding the action of viruses. The list of functional mechanisms in biology that rely on protein–DNA and protein–RNA interactions can be made much longer, but these examples represent some of the topics which motivated structural biologists to study complexes between proteins and nucleic acids as a first step beyond structure determinations of individual biomolecules.
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Breeze, Alexander L. "Isotope-filtered NMR methods for the study of biomolecular structure and interactions." Progress in Nuclear Magnetic Resonance Spectroscopy 36, no. 4 (June 2000): 323–72. http://dx.doi.org/10.1016/s0079-6565(00)00020-0.
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Li, Qingxin, and CongBao Kang. "A Practical Perspective on the Roles of Solution NMR Spectroscopy in Drug Discovery." Molecules 25, no. 13 (June 28, 2020): 2974. http://dx.doi.org/10.3390/molecules25132974.
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Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to study structures and dynamics of biomolecules under physiological conditions. As there are numerous NMR-derived methods applicable to probe protein–ligand interactions, NMR has been widely utilized in drug discovery, especially in such steps as hit identification and lead optimization. NMR is frequently used to locate ligand-binding sites on a target protein and to determine ligand binding modes. NMR spectroscopy is also a unique tool in fragment-based drug design (FBDD), as it is able to investigate target-ligand interactions with diverse binding affinities. NMR spectroscopy is able to identify fragments that bind weakly to a target, making it valuable for identifying hits targeting undruggable sites. In this review, we summarize the roles of solution NMR spectroscopy in drug discovery. We describe some methods that are used in identifying fragments, understanding the mechanism of action for a ligand, and monitoring the conformational changes of a target induced by ligand binding. A number of studies have proven that 19F-NMR is very powerful in screening fragments and detecting protein conformational changes. In-cell NMR will also play important roles in drug discovery by elucidating protein-ligand interactions in living cells.
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Gjuroski, Ilche, Julien Furrer, and Martina Vermathen. "Probing the Interactions of Porphyrins with Macromolecules Using NMR Spectroscopy Techniques." Molecules 26, no. 7 (March 30, 2021): 1942. http://dx.doi.org/10.3390/molecules26071942.
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Porphyrinic compounds are widespread in nature and play key roles in biological processes such as oxygen transport in blood, enzymatic redox reactions or photosynthesis. In addition, both naturally derived as well as synthetic porphyrinic compounds are extensively explored for biomedical and technical applications such as photodynamic therapy (PDT) or photovoltaic systems, respectively. Their unique electronic structures and photophysical properties make this class of compounds so interesting for the multiple functions encountered. It is therefore not surprising that optical methods are typically the prevalent analytical tool applied in characterization and processes involving porphyrinic compounds. However, a wealth of complementary information can be obtained from NMR spectroscopic techniques. Based on the advantage of providing structural and dynamic information with atomic resolution simultaneously, NMR spectroscopy is a powerful method for studying molecular interactions between porphyrinic compounds and macromolecules. Such interactions are of special interest in medical applications of porphyrinic photosensitizers that are mostly combined with macromolecular carrier systems. The macromolecular surrounding typically stabilizes the encapsulated drug and may also modify its physical properties. Moreover, the interaction with macromolecular physiological components needs to be explored to understand and control mechanisms of action and therapeutic efficacy. This review focuses on such non-covalent interactions of porphyrinic drugs with synthetic polymers as well as with biomolecules such as phospholipids or proteins. A brief introduction into various NMR spectroscopic techniques is given including chemical shift perturbation methods, NOE enhancement spectroscopy, relaxation time measurements and diffusion-ordered spectroscopy. How these NMR tools are used to address porphyrin–macromolecule interactions with respect to their function in biomedical applications is the central point of the current review.
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Kolář, Michal H., Gabor Nagy, John Kunkel, Sara M. Vaiana, Lars V. Bock, and Helmut Grubmüller. "Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel." Nucleic Acids Research 50, no. 4 (February 12, 2022): 2258–69. http://dx.doi.org/10.1093/nar/gkac038.
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Abstract The ribosome is a fundamental biomolecular complex that synthesizes proteins in cells. Nascent proteins emerge from the ribosome through a tunnel, where they may interact with the tunnel walls or small molecules such as antibiotics. These interactions can cause translational arrest with notable physiological consequences. Here, we studied the arrest caused by the regulatory peptide VemP, which is known to form α-helices inside the ribosome tunnel near the peptidyl transferase center under specific conditions. We used all-atom molecular dynamics simulations of the entire ribosome and circular dichroism spectroscopy to study the driving forces of helix formation and how VemP causes the translational arrest. To that aim, we compared VemP dynamics in the ribosome tunnel with its dynamics in solution. We show that the VemP peptide has a low helical propensity in water and that the propensity is higher in mixtures of water and trifluorethanol. We propose that helix formation within the ribosome is driven by the interactions of VemP with the tunnel and that a part of VemP acts as an anchor. This anchor might slow down VemP progression through the tunnel enabling α-helix formation, which causes the elongation arrest.
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Hunt, Neil T. "Minding the dynamic gap: measuring ultrafast processes in biomolecular systems." Biochemist 41, no. 2 (April 1, 2019): 30–35. http://dx.doi.org/10.1042/bio04102030.
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Biological processes in vivo involve interactions between large, complex molecules under conditions that are best-approximated by a viscous, multicomponent solution at temperatures often somewhat above room temperature. This means that the molecules involved are dynamic --- their structure changes --- on many timescales in ways that range from very small, localized fluctuations to fundamental changes in their overall appearance. What roles do these dynamics play in the structure–function relationship? Do very fast, local motions have any impact on well-characterized, slower structure changes? How do we go about measuring the very fastest biomolecular fluctuations to find out? Here we discuss ultrafast multidimensional (2D-IR) spectroscopy and look at the complementary information that it provides as part of a raft of biophysical experiments.
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Chakrabarty, Broto, Varun Naganathan, Kanak Garg, Yash Agarwal, and Nita Parekh. "NAPS update: network analysis of molecular dynamics data and protein–nucleic acid complexes." Nucleic Acids Research 47, W1 (May 20, 2019): W462—W470. http://dx.doi.org/10.1093/nar/gkz399.
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Abstract Network theory is now a method of choice to gain insights in understanding protein structure, folding and function. In combination with molecular dynamics (MD) simulations, it is an invaluable tool with widespread applications such as analyzing subtle conformational changes and flexibility regions in proteins, dynamic correlation analysis across distant regions for allosteric communications, in drug design to reveal alternative binding pockets for drugs, etc. Updated version of NAPS now facilitates network analysis of the complete repertoire of these biomolecules, i.e., proteins, protein–protein/nucleic acid complexes, MD trajectories, and RNA. Various options provided for analysis of MD trajectories include individual network construction and analysis of intermediate time-steps, comparative analysis of these networks, construction and analysis of average network of the ensemble of trajectories and dynamic cross-correlations. For protein–nucleic acid complexes, networks of the whole complex as well as that of the interface can be constructed and analyzed. For analysis of proteins, protein–protein complexes and MD trajectories, network construction based on inter-residue interaction energies with realistic edge-weights obtained from standard force fields is provided to capture the atomistic details. Updated version of NAPS also provides improved visualization features, interactive plots and bulk execution. URL: http://bioinf.iiit.ac.in/NAPS/
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Murthy, Anastasia C., and Nicolas L. Fawzi. "The (un)structural biology of biomolecular liquid-liquid phase separation using NMR spectroscopy." Journal of Biological Chemistry 295, no. 8 (January 7, 2020): 2375–84. http://dx.doi.org/10.1074/jbc.rev119.009847.
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Liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a phenomenon that underlies membraneless compartmentalization of the cell. The underlying molecular interactions that underpin biomolecular LLPS have been of increased interest due to the importance of membraneless organelles in facilitating various biological processes and the disease association of several of the proteins that mediate LLPS. Proteins that are able to undergo LLPS often contain intrinsically disordered regions and remain dynamic in solution. Solution-state NMR spectroscopy has emerged as a leading structural technique to characterize protein LLPS due to the variety and specificity of information that can be obtained about intrinsically disordered sequences. This review discusses practical aspects of studying LLPS by NMR, summarizes recent work on the molecular aspects of LLPS of various protein systems, and discusses future opportunities for characterizing the molecular details of LLPS to modulate phase separation.
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Mazurek, Anna Helena, Łukasz Szeleszczuk, Thomas Simonson, and Dariusz Maciej Pisklak. "Application of Various Molecular Modelling Methods in the Study of Estrogens and Xenoestrogens." International Journal of Molecular Sciences 21, no. 17 (September 3, 2020): 6411. http://dx.doi.org/10.3390/ijms21176411.
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In this review, applications of various molecular modelling methods in the study of estrogens and xenoestrogens are summarized. Selected biomolecules that are the most commonly chosen as molecular modelling objects in this field are presented. In most of the reviewed works, ligand docking using solely force field methods was performed, employing various molecular targets involved in metabolism and action of estrogens. Other molecular modelling methods such as molecular dynamics and combined quantum mechanics with molecular mechanics have also been successfully used to predict the properties of estrogens and xenoestrogens. Among published works, a great number also focused on the application of different types of quantitative structure–activity relationship (QSAR) analyses to examine estrogen’s structures and activities. Although the interactions between estrogens and xenoestrogens with various proteins are the most commonly studied, other aspects such as penetration of estrogens through lipid bilayers or their ability to adsorb on different materials are also explored using theoretical calculations. Apart from molecular mechanics and statistical methods, quantum mechanics calculations are also employed in the studies of estrogens and xenoestrogens. Their applications include computation of spectroscopic properties, both vibrational and Nuclear Magnetic Resonance (NMR), and also in quantum molecular dynamics simulations and crystal structure prediction. The main aim of this review is to present the great potential and versatility of various molecular modelling methods in the studies on estrogens and xenoestrogens.
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Boelens, Rolf, Konstantin Ivanov, and Jörg Matysik. "Introduction to a special issue of <i>Magnetic Resonance</i> in honour of Robert Kaptein at the occasion of his 80th birthday." Magnetic Resonance 2, no. 1 (June 17, 2021): 465–74. http://dx.doi.org/10.5194/mr-2-465-2021.
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Abstract. This publication, in honour of Robert Kaptein's 80th birthday, contains contributions from colleagues, many of whom have worked with him, and others who admire his work and have been stimulated by his research. The contributions show current research in biomolecular NMR, spin hyperpolarisation and spin chemistry, including CIDNP (chemically induced dynamic nuclear polarisation), topics to which he has contributed enormously. His proposal of the radical pair mechanism was the birth of the field of spin chemistry, and the laser CIDNP NMR experiment on a protein was a major breakthrough in hyperpolarisation research. He set milestones for biomolecular NMR by developing computational methods for protein structure determination, including restrained molecular dynamics and 3D NMR methodology. With a lac repressor headpiece, he determined one of the first protein structures determined by NMR. His studies of the lac repressor provided the first examples of detailed studies of protein nucleic acid complexes by NMR. This deepened our understanding of protein DNA recognition and led to a molecular model for protein sliding along the DNA. Furthermore, he played a leading role in establishing the cluster of NMR large-scale facilities in Europe. This editorial gives an introduction to the publication and is followed by a biography describing his contributions to magnetic resonance.
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Nguyen, Trang Thi Thuy, Seungjoo Haam, Joon-Seo Park, and Sang-Wha Lee. "Cysteine-Encapsulated Liposome for Investigating Biomolecular Interactions at Lipid Membranes." International Journal of Molecular Sciences 23, no. 18 (September 12, 2022): 10566. http://dx.doi.org/10.3390/ijms231810566.
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The development of a strategy to investigate interfacial phenomena at lipid membranes is practically useful because most essential biomolecular interactions occur at cell membranes. In this study, a colorimetric method based on cysteine-encapsulated liposomes was examined using gold nanoparticles as a probe to provide a platform to report an enzymatic activity at lipid membranes. The cysteine-encapsulated liposomes were prepared with varying ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol through the hydration of lipid films and extrusions in the presence of cysteine. The size, composition, and stability of resulting liposomes were analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS), nuclear magnetic resonance (NMR) spectroscopy, and UV-vis spectrophotometry. The results showed that the increased cholesterol content improved the stability of liposomes, and the liposomes were formulated with 60 mol % cholesterol for the subsequent experiments. Triton X-100 was tested to disrupt the lipid membranes to release the encapsulated cysteine from the liposomes. Cysteine can induce the aggregation of gold nanoparticles accompanying a color change, and the colorimetric response of gold nanoparticles to the released cysteine was investigated in various media. Except in buffer solutions at around pH 5, the cysteine-encapsulated liposomes showed the color change of gold nanoparticles only after being incubated with Triton X-100. Finally, the cysteine-encapsulated liposomal platform was tested to report the enzymatic activity of phospholipase A2 that hydrolyzes phospholipids in the membrane. The hydrolysis of phospholipids triggered the release of cysteine from the liposomes, and the released cysteine was successfully detected by monitoring the distinct red-to-blue color change of gold nanoparticles. The presence of phospholipase A2 was also confirmed by the appearance of a peak around 690 nm in the UV-vis spectra, which is caused by the cysteine-induced aggregation of gold nanoparticles. The results demonstrated that the cysteine-encapsulated liposome has the potential to be used to investigate biological interactions occurring at lipid membranes.
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Pasquali, S., E. Frezza, and F. L. Barroso da Silva. "Coarse-grained dynamic RNA titration simulations." Interface Focus 9, no. 3 (April 19, 2019): 20180066. http://dx.doi.org/10.1098/rsfs.2018.0066.
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Electrostatic interactions play a pivotal role in many biomolecular processes. The molecular organization and function in biological systems are largely determined by these interactions. Owing to the highly negative charge of RNA, the effect is expected to be more pronounced in this system. Moreover, RNA base pairing is dependent on the charge of the base, giving rise to alternative secondary and tertiary structures. The equilibrium between uncharged and charged bases is regulated by the solution pH, which is therefore a key environmental condition influencing the molecule’s structure and behaviour. By means of constant-pH Monte Carlo simulations based on a fast proton titration scheme, coupled with the coarse-grained model HiRE-RNA, molecular dynamic simulations of RNA molecules at constant pH enable us to explore the RNA conformational plasticity at different pH values as well as to compute electrostatic properties as local p K a values for each nucleotide.
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Kuschert, Sarah, Martin Stroet, Yanni Ka-Yan Chin, Anne Claire Conibear, Xinying Jia, Thomas Lee, Christian Reinhard Otto Bartling, et al. "Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR." Magnetic Resonance 4, no. 1 (February 24, 2023): 57–72. http://dx.doi.org/10.5194/mr-4-57-2023.
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Abstract. Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (https://atb.uq.edu.au, last access: 17 February 2023) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of side chains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice, we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein–protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic biology and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally.
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Pomin, Vitor, and Xu Wang. "Glycosaminoglycan-Protein Interactions by Nuclear Magnetic Resonance (NMR) Spectroscopy." Molecules 23, no. 9 (September 11, 2018): 2314. http://dx.doi.org/10.3390/molecules23092314.
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Nuclear magnetic resonance (NMR) spectroscopy is one of the most utilized and informative analytical techniques for investigating glycosaminoglycan (GAG)-protein complexes. NMR methods that are commonly applied to GAG-protein systems include chemical shift perturbation, saturation transfer difference, and transferred nuclear Overhauser effect. Although these NMR methods have revealed valuable insight into the protein-GAG complexes, elucidating high-resolution structural and dynamic information of these often transient interactions remains challenging. In addition, preparation of structurally homogeneous and isotopically enriched GAG ligands for structural investigations continues to be laborious. As a result, understanding of the structure-activity relationship of GAGs is still primitive. To overcome these deficiencies, several innovative NMR techniques have been developed lately. Here, we review some of the commonly used techniques along with more novel methods such as waterLOGSY and experiments to examine structure and dynamic of lysine and arginine side chains to identify GAG-binding sites. We will also present the latest technology that is used to produce isotopically enriched as well as paramagnetically tagged GAG ligands. Recent results that were obtained from solid-state NMR of amyloid’s interaction with GAG are also presented together with a brief discussion on computer assisted modeling of GAG-protein complexes using sparse experimental data.
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Krishnan, Viswanathan. "Molecular Thermodynamics Using Nuclear Magnetic Resonance (NMR) Spectroscopy." Inventions 4, no. 1 (February 21, 2019): 13. http://dx.doi.org/10.3390/inventions4010013.
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Nuclear magnetic resonance (NMR) spectroscopy is perhaps the most widely used technology from the undergraduate teaching labs in organic chemistry to advanced research for the determination of three-dimensional structure as well as dynamics of biomolecular systems... The NMR spectrum of a molecule under a given experimental condition is unique, providing both quantitative and structural information. In particular, the quantitative nature of NMR spectroscopy offers the ability to follow a reaction pathway of the given molecule in a dynamic process under well-defined experimental conditions. To highlight the use of NMR when determining the molecular thermodynamic parameters, a review of three distinct applications developed from our laboratory is presented. These applications include the thermodynamic parameters of (a) molecular oxidation from time-dependent kinetics, (b) intramolecular rotation, and (c) intermolecular exchange. An experimental overview and the method of data analysis are provided so that these applications can be adopted in a range of molecular systems.
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Moore, James T., Nicholas E. Smith, and Connie C. Lu. "Structure and dynamic NMR behavior of rhodium complexes supported by Lewis acidic group 13 metallatranes." Dalton Transactions 46, no. 17 (2017): 5689–701. http://dx.doi.org/10.1039/c6dt04769f.
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Paczwa, Mateusz, Aleksej A. Sapiga, Marcin Olszewski, Nikolaj Sergeev, and Aleksej V. Sapiga. "23Na Nuclear Magnetic Resonance Study of the Structure and Dynamic of Natrolite." Zeitschrift für Naturforschung A 70, no. 4 (April 1, 2015): 295–300. http://dx.doi.org/10.1515/zna-2014-0371.
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AbstractThe temperature dependences of nuclear magnetic resonance (NMR) and magic angle spinning (MAS) NMR spectra of 23Na nuclei in natrolite (Na2Al2Si3O10·2H2O) have been studied. The temperature dependences of the spin-lattice relaxation times T1 in natrolite have also been studied. It has been shown that the spin-lattice relaxation of the 23Na is governed by the electric quadrupole interaction with the crystal electric field gradients modulated by translational motion of H2O molecules in the natrolite pores. The dipolar interactions with paramagnetic impurities become significant as a relaxation mechanism of the 23Na nuclei only at low temperature (<270 K).
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Kumar, Akshita, Harini Mohanram, Kiat Whye Kong, Rubayn Goh, Shawn Hoon, Julien Lescar, and Ali Miserez. "Supramolecular propensity of suckerin proteins is driven by β-sheets and aromatic interactions as revealed by solution NMR." Biomaterials Science 6, no. 9 (2018): 2440–47. http://dx.doi.org/10.1039/c8bm00556g.
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Yeh, Vivien, Alice Goode, and Boyan B. Bonev. "Membrane Protein Structure Determination and Characterisation by Solution and Solid-State NMR." Biology 9, no. 11 (November 12, 2020): 396. http://dx.doi.org/10.3390/biology9110396.
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Biological membranes define the interface of life and its basic unit, the cell. Membrane proteins play key roles in membrane functions, yet their structure and mechanisms remain poorly understood. Breakthroughs in crystallography and electron microscopy have invigorated structural analysis while failing to characterise key functional interactions with lipids, small molecules and membrane modulators, as well as their conformational polymorphism and dynamics. NMR is uniquely suited to resolving atomic environments within complex molecular assemblies and reporting on membrane organisation, protein structure, lipid and polysaccharide composition, conformational variations and molecular interactions. The main challenge in membrane protein studies at the atomic level remains the need for a membrane environment to support their fold. NMR studies in membrane mimetics and membranes of increasing complexity offer close to native environments for structural and molecular studies of membrane proteins. Solution NMR inherits high resolution from small molecule analysis, providing insights from detergent solubilised proteins and small molecular assemblies. Solid-state NMR achieves high resolution in membrane samples through fast sample spinning or sample alignment. Recent developments in dynamic nuclear polarisation NMR allow signal enhancement by orders of magnitude opening new opportunities for expanding the applications of NMR to studies of native membranes and whole cells.
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Lecoq, Lauriane, Marie-Laure Fogeron, Beat H. Meier, Michael Nassal, and Anja Böckmann. "Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies." Viruses 12, no. 10 (September 24, 2020): 1069. http://dx.doi.org/10.3390/v12101069.
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Structural virology reveals the architecture underlying infection. While notably electron microscopy images have provided an atomic view on viruses which profoundly changed our understanding of these assemblies incapable of independent life, spectroscopic techniques like NMR enter the field with their strengths in detailed conformational analysis and investigation of dynamic behavior. Typically, the large assemblies represented by viral particles fall in the regime of biological high-resolution solid-state NMR, able to follow with high sensitivity the path of the viral proteins through their interactions and maturation steps during the viral life cycle. We here trace the way from first solid-state NMR investigations to the state-of-the-art approaches currently developing, including applications focused on HIV, HBV, HCV and influenza, and an outlook to the possibilities opening in the coming years.
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Chroni, Angeliki, Thomas Mavromoustakos, and Stergios Pispas. "Biocompatible PEO-b-PCL Nanosized Micelles as Drug Carriers: Structure and Drug–Polymer Interactions." Nanomaterials 10, no. 9 (September 18, 2020): 1872. http://dx.doi.org/10.3390/nano10091872.
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We report on the preparation of drug nanocarriers by encapsulating losartan potassium (LSR) into amphiphilic block copolymer micelles, utilizing the biocompatible/biodegradable poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) diblock copolymer. The PEO-b-PCL micelles and LSR-loaded PEO-b-PCL nanocarriers were prepared by organic solvent evaporation method (OSEM). Light scattering and nuclear magnetic resonance (NMR) provide information on micelle structure and polymer–drug interactions. According to dynamic light scattering (DLS) analysis, the PEO-b-PCL micelles and LSR-loaded PEO-b-PCL nanocarriers formed nanostructures in the range of 17–26 nm in aqueous milieu. Attenuated total reflection Fourier transform infrared (ATR-FTIR) and ultraviolet-visible (UV-Vis) measurements confirmed the presence of LSR in the polymeric drug solutions. NMR results proved the successful encapsulation of LSR into the PEO-b-PCL micelles by analyzing the drug–micelles intermolecular interactions. Specifically, 2D-NOESY experiments clearly evidenced the intermolecular interactions between the biphenyl ring and butyl chain of LSR structure with the methylene signals of PCL. Additionally, NMR studies as a function of temperature demonstrated an unexpected, enhanced proton mobility of the PEO-b-PCL micellar core in D2O solutions, probably caused by the melting of the PCL hydrophobic core.
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Thomas, John J., Brian Bothner, Joe Traina, W. Henry Benner, and Gary Siuzdak. "Electrospray ion mobility spectrometry of intact viruses." Spectroscopy 18, no. 1 (2004): 31–36. http://dx.doi.org/10.1155/2004/376572.
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Characterizing supramolecular interactions offers significant challenges using NMR or crystallographic techniques either because of size limitations or the difficulty in forming suitable crystals, while mass spectrometry is largely limited to low resolution mass information. Here we report gas phase measurements of intact virus particles using electrospray ion mobility spectrometry with an accuracy in radial measurements that were sufficient to differentiate closely related species. In addition, measured diameters indicate that iscosahedral virus particles retain their structure in the gas phase as well as undergoing a slight compaction in the absence of solvent. Analysis of the human pathogen adenovirus represents the largest and most sophisticated biomolecular complex detected in the gas phase to date. These results, on a diverse set of viral systems, suggest that ion mobility spectrometry may have broad applications for the analysis of biological complexes.
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Ferro, Monica, Franca Castiglione, Nadia Pastori, Carlo Punta, Lucio Melone, Walter Panzeri, Barbara Rossi, Francesco Trotta, and Andrea Mele. "Dynamics and interactions of ibuprofen in cyclodextrin nanosponges by solid-state NMR spectroscopy." Beilstein Journal of Organic Chemistry 13 (January 27, 2017): 182–94. http://dx.doi.org/10.3762/bjoc.13.21.
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Two different formulations of cyclodextrin nanosponges (CDNS), obtained by polycondensation of β-cyclodextrin with ethylenediaminetetraacetic acid dianhydride (EDTAn), were treated with aqueous solutions of ibuprofen sodium salt (IbuNa) affording hydrogels that, after lyophilisation, gave two solid CDNS-drug formulations. 1H fast MAS NMR and 13C CP-MAS NMR spectra showed that IbuNa was converted in situ into its acidic and dimeric form (IbuH) after freeze-drying. 13C CP-MAS NMR spectra also indicated that the structure of the nanosponge did not undergo changes upon drug loading compared to the unloaded system. However, the 13C NMR spectra collected under variable contact time cross-polarization (VCT-CP) conditions showed that the polymeric scaffold CDNS changed significantly its dynamic regime on passing from the empty CDNS to the drug-loaded CDNS, thus showing that the drug encapsulation can be seen as the formation of a real supramolecular aggregate rather than a conglomerate of two solid components. Finally, the structural features obtained from the different solid-state NMR approaches reported matched the information from powder X-ray diffraction profiles.
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Huang, Yen-Hua, and Cheng-Yang Huang. "Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT." BioMed Research International 2014 (2014): 1–14. http://dx.doi.org/10.1155/2014/195162.
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Replication restart primosome is a complex dynamic system that is essential for bacterial survival. This system uses various proteins to reinitiate chromosomal DNA replication to maintain genetic integrity after DNA damage. The replication restart primosome inEscherichia coliis composed of PriA helicase, PriB, PriC, DnaT, DnaC, DnaB helicase, and DnaG primase. The assembly of the protein complexes within the forked DNA responsible for reloading the replicative DnaB helicase anywhere on the chromosome for genome duplication requires the coordination of transient biomolecular interactions. Over the last decade, investigations on the structure and mechanism of these nucleoproteins have provided considerable insight into primosome assembly. In this review, we summarize and discuss our current knowledge and recent advances on the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.
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Helliwell, John R., Alice Brink, Surasak Kaenket, Victoria Laurina Starkey, and Simon W. M. Tanley. "X-ray diffraction in temporally and spatially resolved biomolecular science." Faraday Discussions 177 (2015): 429–41. http://dx.doi.org/10.1039/c4fd00166d.
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Time-resolved Laue protein crystallography at the European Synchrotron Radiation Facility (ESRF) opened up the field of sub-nanosecond protein crystal structure analyses. There are a limited number of such time-resolved studies in the literature. Why is this? The X-ray laser now gives us femtosecond (fs) duration pulses, typically 10 fs up to ∼50 fs. Their use is attractive for the fastest time-resolved protein crystallography studies. It has been proposed that single molecules could even be studied with the advantage of being able to measure X-ray diffraction from a ‘crystal lattice free’ single molecule, with or without temporal resolved structural changes. This is altogether very challenging R&D. So as to assist this effort we have undertaken studies of metal clusters that bind to proteins, both ‘fresh’ and after repeated X-ray irradiation to assess their X-ray-photo-dynamics, namely Ta6Br12, K2PtI6 and K2PtBr6 bound to a test protein, hen egg white lysozyme. These metal complexes have the major advantage of being very recognisable shapes (pseudo spherical or octahedral) and thereby offer a start to (probably very difficult) single molecule electron density map interpretations, both static and dynamic. A further approach is to investigate the X-ray laser beam diffraction strength of a well scattering nano-cluster; an example from nature being the iron containing ferritin. Electron crystallography and single particle electron microscopy imaging offers alternatives to X-ray structural studies; our structural studies of crustacyanin, a 320 kDa protein carotenoid complex, can be extended either by electron based techniques or with the X-ray laser representing a fascinating range of options. General outlook remarks concerning X-ray, electron and neutron macromolecular crystallography as well as ‘NMR crystallography’ conclude the article.
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Agback, Tatiana, Francisco Dominguez, Ilya Frolov, Elena I. Frolova, and Peter Agback. "1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution." PLOS ONE 16, no. 12 (December 7, 2021): e0251834. http://dx.doi.org/10.1371/journal.pone.0251834.
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Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the Cβ of the apo form of the full-lengthSARS-CoV-2 nsp1 including the folded domain together with the flaking N- and C- terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure as well as another published secondary structure obtained by NMR, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.
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Mittag, Tanja, Stephen Orlicky, Wing-Yiu Choy, Xiaojing Tang, Hong Lin, Frank Sicheri, Lewis E. Kay, Mike Tyers, and Julie D. Forman-Kay. "Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor." Proceedings of the National Academy of Sciences 105, no. 46 (November 13, 2008): 17772–77. http://dx.doi.org/10.1073/pnas.0809222105.
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Intrinsically disordered proteins play critical but often poorly understood roles in mediating protein interactions. The interactions of disordered proteins studied to date typically entail structural stabilization, whether as a global disorder-to-order transition or minimal ordering of short linear motifs. The disordered cyclin-dependent kinase (CDK) inhibitor Sic1 interacts with a single site on its receptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The molecular basis for this multisite-dependent interaction with a single receptor site is not known. By NMR analysis, we show that multiple phosphorylated sites on Sic1 interact with Cdc4 in dynamic equilibrium with only local ordering around each site. Regardless of phosphorylation status, Sic1 exists in an intrinsically disordered state but is surprisingly compact with transient structure. The observation of this unusual binding mode between Sic1 and Cdc4 extends the understanding of protein interactions from predominantly static complexes to include dynamic ensembles of intrinsically disordered states.
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Jeon, Jaekyun, Kent R. Thurber, Rodolfo Ghirlando, Wai-Ming Yau, and Robert Tycko. "Application of millisecond time-resolved solid state NMR to the kinetics and mechanism of melittin self-assembly." Proceedings of the National Academy of Sciences 116, no. 34 (August 6, 2019): 16717–22. http://dx.doi.org/10.1073/pnas.1908006116.
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Common experimental approaches for characterizing structural conversion processes such as protein folding and self-assembly do not report on all aspects of the evolution from an initial state to the final state. Here, we demonstrate an approach that is based on rapid mixing, freeze-trapping, and low-temperature solid-state NMR (ssNMR) with signal enhancements from dynamic nuclear polarization (DNP). Experiments on the folding and tetramerization of the 26-residue peptide melittin following a rapid pH jump show that multiple aspects of molecular structure can be followed with millisecond time resolution, including secondary structure at specific isotopically labeled sites, intramolecular and intermolecular contacts between specific pairs of labeled residues, and overall structural order. DNP-enhanced ssNMR data reveal that conversion of conformationally disordered melittin monomers at low pH to α-helical conformations at neutral pH occurs on nearly the same timescale as formation of antiparallel melittin dimers, about 6 to 9 ms for 0.3 mM melittin at 24 °C in aqueous solution containing 20% (vol/vol) glycerol and 75 mM sodium phosphate. Although stopped-flow fluorescence data suggest that melittin tetramers form quickly after dimerization, ssNMR spectra show that full structural order within melittin tetramers develops more slowly, in ∼60 ms. Time-resolved ssNMR is likely to find many applications to biomolecular structural conversion processes, including early stages of amyloid formation, viral capsid formation, and protein–protein recognition.
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Marques-Magalhães, Ângela, Tânia Cruz, Ângela Margarida Costa, Diogo Estêvão, Elisabete Rios, Pedro Amoroso Canão, Sérgia Velho, Fátima Carneiro, Maria José Oliveira, and Ana Patrícia Cardoso. "Decellularized Colorectal Cancer Matrices as Bioactive Scaffolds for Studying Tumor-Stroma Interactions." Cancers 14, no. 2 (January 12, 2022): 359. http://dx.doi.org/10.3390/cancers14020359.
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More than a physical structure providing support to tissues, the extracellular matrix (ECM) is a complex and dynamic network of macromolecules that modulates the behavior of both cancer cells and associated stromal cells of the tumor microenvironment (TME). Over the last few years, several efforts have been made to develop new models that accurately mimic the interconnections within the TME and specifically the biomechanical and biomolecular complexity of the tumor ECM. Particularly in colorectal cancer, the ECM is highly remodeled and disorganized and constitutes a key component that affects cancer hallmarks, such as cell differentiation, proliferation, angiogenesis, invasion and metastasis. Therefore, several scaffolds produced from natural and/or synthetic polymers and ceramics have been used in 3D biomimetic strategies for colorectal cancer research. Nevertheless, decellularized ECM from colorectal tumors is a unique model that offers the maintenance of native ECM architecture and molecular composition. This review will focus on innovative and advanced 3D-based models of decellularized ECM as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro 2D and in vivo models. Our aim is to highlight the need for strategies that accurately mimic the TME for precision medicine and for studying the pathophysiology of the disease.
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Rajesh, Sundaresan, Pooja Sridhar, Birke Andrea Tews, Lucie Fénéant, Laurence Cocquerel, Douglas G. Ward, Fedor Berditchevski, and Michael Overduin. "Structural Basis of Ligand Interactions of the Large Extracellular Domain of Tetraspanin CD81." Journal of Virology 86, no. 18 (June 27, 2012): 9606–16. http://dx.doi.org/10.1128/jvi.00559-12.
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Hepatitis C virus (HCV) causes chronic liver disease, cirrhosis, and primary liver cancer. Despite 130 million people being at risk worldwide, no vaccine exists, and effective therapy is limited by drug resistance, toxicity, and high costs. The tetraspanin CD81 is an essential entry-level receptor required for HCV infection of hepatocytes and represents a critical target for intervention. In this study, we report the first structural characterization of the large extracellular loop of CD81, expressed in mammalian cells and studied in physiological solutions. The HCV E2 glycoprotein recognizes CD81 through a dynamic loop on the helical bundle, which was shown by nuclear magnetic resonance (NMR) spectroscopy to adopt a conformation distinct from that seen in crystals. A novel membrane binding interface was revealed adjacent to the exposed HCV interaction site in the extracellular loop of CD81. The binding pockets for two proposed inhibitors of the CD81-HCV interaction, namely, benzyl salicylate and fexofenadine, were shown to overlap the HCV and membrane interaction sites. Although the dynamic loop region targeted by these compounds presents challenges for structure-based design, the NMR assignments enable realistic screening and validation of ligands. Together, these data provide an improved avenue for developing potent agents that specifically block CD81-HCV interaction and also pave a way for elucidating the recognition mechanisms of diverse tetraspanins.
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Lange, Sascha, W. Trent Franks, Nandhakishore Rajagopalan, Kristina Döring, Michel A. Geiger, Arne Linden, Barth-Jan van Rossum, Günter Kramer, Bernd Bukau, and Hartmut Oschkinat. "Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR." Science Advances 2, no. 8 (August 2016): e1600379. http://dx.doi.org/10.1126/sciadv.1600379.
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Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA–adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.
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Mollner, Tim A., Patrick G. Isenegger, Brian Josephson, Charles Buchanan, Lukas Lercher, Daniel Oehlrich, D. Flemming Hansen, et al. "Post-translational insertion of boron in proteins to probe and modulate function." Nature Chemical Biology 17, no. 12 (November 1, 2021): 1245–61. http://dx.doi.org/10.1038/s41589-021-00883-7.
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AbstractBoron is absent in proteins, yet is a micronutrient. It possesses unique bonding that could expand biological function including modes of Lewis acidity not available to typical elements of life. Here we show that post-translational Cβ–Bγ bond formation provides mild, direct, site-selective access to the minimally sized residue boronoalanine (Bal) in proteins. Precise anchoring of boron within complex biomolecular systems allows dative bond-mediated, site-dependent protein Lewis acid–base-pairing (LABP) by Bal. Dynamic protein-LABP creates tunable inter- and intramolecular ligand–host interactions, while reactive protein-LABP reveals reactively accessible sites through migratory boron-to-oxygen Cβ–Oγ covalent bond formation. These modes of dative bonding can also generate de novo function, such as control of thermo- and proteolytic stability in a target protein, or observation of transient structural features via chemical exchange. These results indicate that controlled insertion of boron facilitates stability modulation, structure determination, de novo binding activities and redox-responsive ‘mutation’.
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Bottaro, Sandro, Parker J. Nichols, Beat Vögeli, Michele Parrinello, and Kresten Lindorff-Larsen. "Integrating NMR and simulations reveals motions in the UUCG tetraloop." Nucleic Acids Research 48, no. 11 (May 19, 2020): 5839–48. http://dx.doi.org/10.1093/nar/gkaa399.
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Abstract We provide an atomic-level description of the structure and dynamics of the UUCG RNA stem–loop by combining molecular dynamics simulations with experimental data. The integration of simulations with exact nuclear Overhauser enhancements data allowed us to characterize two distinct states of this molecule. The most stable conformation corresponds to the consensus three-dimensional structure. The second state is characterized by the absence of the peculiar non-Watson–Crick interactions in the loop region. By using machine learning techniques we identify a set of experimental measurements that are most sensitive to the presence of non-native states. We find that although our MD ensemble, as well as the consensus UUCG tetraloop structures, are in good agreement with experiments, there are remaining discrepancies. Together, our results show that (i) the MD simulation overstabilize a non-native loop conformation, (ii) eNOE data support its presence with a population of ≈10% and (iii) the structural interpretation of experimental data for dynamic RNAs is highly complex, even for a simple model system such as the UUCG tetraloop.
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Perry, Alexis, and Christina J. Kousseff. "Synthesis and metal binding properties of N-alkylcarboxyspiropyrans." Beilstein Journal of Organic Chemistry 13 (August 4, 2017): 1542–50. http://dx.doi.org/10.3762/bjoc.13.154.
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Spiropyrans bearing an N-alkylcarboxylate tether are a common structure in dynamic, photoactive materials and serve as colourimetric/fluorimetric cation receptors. In this study, we describe an efficient synthesis of spiropyrans with 2–12 carbon atom alkylcarboxylate substituents, and a systematic analysis of their interactions with metal cations using 1H NMR and UV-visible spectroscopy. All N-alkylcarboxyspiropyrans in this study displayed a strong preference for binding divalent metal cations and a modest increase in M2+ binding affinity correlated with increased alkycarboxylate tether length.
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Metcalf, Douglas G., Joseph M. Kielec, Kathleen G. Valentine, A. Joshua Wand, William F. DeGrado, and Joel S. Bennett. "NMR Structure of a Disulfide-Crosslinked αIIbβ3 Cytoplasmic Domain Heterodimer." Blood 112, no. 11 (November 16, 2008): 2866. http://dx.doi.org/10.1182/blood.v112.11.2866.2866.
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Abstract The platelet integrin αIIbβ3 is the prototypic example of regulated integrin function. Thus, αIIbβ3 is present in a resting conformation on unstimulated platelets, but switches to an active conformation following platelet stimulation. Recent experiments suggest that disrupting a heteromeric interaction between the αIIb and β3 transmembrane (TM) and cytoplasmic domains shifts αIIbβ3 from its resting to its active conformation. However, structural information about the heteromeric interaction is sparse. Thus far, the structure of the TM heterodimer has only been studied by molecular modeling. Interactions between soluble cytosolic tail peptides have been studied by NMR spectroscopy, but these studies may not reflect native contacts because they fail to account for constraining TM domain interactions. To obtain an NMR structure for the αIIbβ3 cytosolic tail heterodimer that reflects its native structure, we expressed 13C- and 15N-labeled peptides corresponding to αIIb residues 988–1008 and β3 residues 713–762 in E. coli. Residues 987 in αIIb and 712 in β3 were replaced with cysteines, based on modeling that predicts the resultant disulfide bond will fix the peptides in their native orientation. Crosslinked heterodimers were dissolved in dodecylphosphocholine micelles at pH 6.5 and analyzed at 37°C on a 750 MHz NMR spectrometer. Previously, we presented a preliminary analysis of this construct indicating that when constrained by the proximal disulfide bond, the αIIb and β3 cytoplasmic tails interact and the cytosolic tail of β3 consists of three helices. We have now solved the final structure which defines the β3 interface that interacts with the αIIb cytoplasmic tail. The αIIb-β3 heterodimer interface is dynamic, but can be localized to β3 residues 716 and 719 because they have different chemical shifts in the crosslinked heterodimer than they do in the component monomers. This positions β3 residue 723 at the αIIb-β3 interface, consistent with the putative Arg995-Asp723 salt bridge. Interestingly, the αIIb tail is natively unstructured so a static interface for αIIb could not be identified. Additionally, the completed structure defines the relative orientations of the three β3 helices. The β3 cytoplasmic tail contains a sharp kink at residue 724 that fixes the membrane embedded helix (residues 713–723) and the first cytoplasmic helix (residues 725–736) at a right angle. The kink was defined by multiple NMR parameters including NOE distance restraints between residues 721 and 727. The distal cytoplasmic helix (residues 746–757) is related to the rest of the molecule by a flexible loop (residues 737– 745). N15 NOESY-HSQC crosspeak intensities provide evidence that the flexible loop and distal helix undergo increased motion relative to the first two helices, and the final structure reflects this motion because there is no preferred orientation for the distal helix relative to the first two helices. Lastly, the distal helix and flexible loop are joined by β3’s canonical NPXY motif which forms an N-terminal cap for the distal helix. In conclusion, we have solved the NMR structure of a disulfide-crosslinked αIIb/β3 cytoplasmic tail heterodimer. Our analysis indicates that, when constrained by a disulfide bond, the αIIb and β3 cytoplasmic tails interact, providing one mechanism for maintaining αIIbβ3 in a resting state.
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Karamanos, Theodoros K., Vitali Tugarinov, and G. Marius Clore. "Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis." Proceedings of the National Academy of Sciences 116, no. 43 (October 7, 2019): 21529–38. http://dx.doi.org/10.1073/pnas.1914999116.
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J-domain chaperones are involved in the efficient handover of misfolded/partially folded proteins to Hsp70 but also function independently to protect against cell death. Due to their high flexibility, the mechanism by which they regulate the Hsp70 cycle and how specific substrate recognition is performed remains unknown. Here we focus on DNAJB6b, which has been implicated in various human diseases and represents a key player in protection against neurodegeneration and protein aggregation. Using a variant that exists mainly in a monomeric form, we report the solution structure of an Hsp40 containing not only the J and C-terminal substrate binding (CTD) domains but also the functionally important linkers. The structure reveals a highly dynamic protein in which part of the linker region masks the Hsp70 binding site. Transient interdomain interactions via regions crucial for Hsp70 binding create a closed, autoinhibited state and help retain the monomeric form of the protein. Detailed NMR analysis shows that the CTD (but not the J domain) self-associates to form an oligomer comprising ∼35 monomeric units, revealing an intricate balance between intramolecular and intermolecular interactions. The results shed light on the mechanism of autoregulation of the Hsp70 cycle via conserved parts of the linker region and reveal the mechanism of DNAJB6b oligomerization and potentially antiaggregation.
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Kleinpeter, Erich. "Push-pull alkenes: Structure and -electron distribution." Journal of the Serbian Chemical Society 71, no. 1 (2006): 1–17. http://dx.doi.org/10.2298/jsc0601001k.
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Push-pull alkenes are substituted alkenes with one or two electron-donating substituents on one end of C=C double bond and with one or two electron-accepting substituents at the other end. Allowance for ?-electron delocalization leads to the central C=C double bond becoming ever more polarized and with rising push-pull character, the ?-bond order of this double bond is reduced and, conversely, the corresponding ?-bond orders of the C-Don and C-Acc bonds are accordingly increased. This push-pull effect is of decisive influence on both the dynamic behavior and the chemical reactivity of this class of compounds and thus it is of considerable interest to both determine and to quantify the inherent push-pull effect. Previously, the barriers to rotation about the C=C, C-Don and/or C-Acc partial double bonds (?G?, as determined by dynamic NMR spectroscopy) or the 13C chemical shift difference of the polarized C=C partial double bond (??C=C) were employed for this purpose. However, these parameters can have serious limitations, viz. the barriers can be immeasurable on the NMR timescale (either by being too high or too low; heavily-biased conformers are present, etc.) or ??C=C behaves in a non-additive manner with respect to the combination of the four substituents. Hence, a general parameter to quantify the push-pull effect is not yet available. Ab initio MO calculations on a collection of compounds, together with NBO analysis, provided valuable information on the structure, bond energies, electron occupancies and bonding/antibonding interactions. In addition to ?G?C=C (either experimentally determined or theoretically calculated) and ??C=C, the bond length of the C=C partial double bond was also examined and it proved to be a reliable parameter to quantify the push-pull effect. Equally so, the quotient of the occupation numbers of the antibonding and bonding ? orbitals of the central C=C partial double bond ( ?*C=C/ ?C=C) could also be employed for this purpose. .