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1
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 (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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Garaizar, Adiran, Ignacio Sanchez-Burgos, Rosana Collepardo-Guevara, and Jorge R. Espinosa. "Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid–Liquid Phase Separation." Molecules 25, no. 20 (2020): 4705. http://dx.doi.org/10.3390/molecules25204705.
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Proteins containing intrinsically disordered regions (IDRs) are ubiquitous within biomolecular condensates, which are liquid-like compartments within cells formed through liquid–liquid phase separation (LLPS). The sequence of amino acids of a protein encodes its phase behaviour, not only by establishing the patterning and chemical nature (e.g., hydrophobic, polar, charged) of the various binding sites that facilitate multivalent interactions, but also by dictating the protein conformational dynamics. Besides behaving as random coils, IDRs can exhibit a wide-range of structural behaviours, including conformational switching, where they transition between alternate conformational ensembles. Using Molecular Dynamics simulations of a minimal coarse-grained model for IDRs, we show that the role of protein conformation has a non-trivial effect in the liquid–liquid phase behaviour of IDRs. When an IDR transitions to a conformational ensemble enriched in disordered extended states, LLPS is enhanced. In contrast, IDRs that switch to ensembles that preferentially sample more compact and structured states show inhibited LLPS. This occurs because extended and disordered protein conformations facilitate LLPS-stabilising multivalent protein–protein interactions by reducing steric hindrance; thereby, such conformations maximize the molecular connectivity of the condensed liquid network. Extended protein configurations promote phase separation regardless of whether LLPS is driven by homotypic and/or heterotypic protein–protein interactions. This study sheds light on the link between the dynamic conformational plasticity of IDRs and their liquid–liquid phase behaviour.
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Mizutani, Tadashi, and Shigeyuki Yagi. "Linear tetrapyrroles as functional pigments in chemistry and biology." Journal of Porphyrins and Phthalocyanines 08, no. 03 (2004): 226–37. http://dx.doi.org/10.1142/s1088424604000210.
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1,19,21,24-tetrahydro-1,19-bilindione is the framework of pigments frequently found in nature, which includes biliverdin IX α, phytochromobilin and phycocyanobilin. 1,19-bilindiones have unique features such as (1) photochemical and thermal cis-trans isomerization, (2) excited energy transfer, (3) chiroptical properties due to the cyclic helical conformation, (4) redox activity, (5) coordination to various metals, and (6) reconstitution to proteins. 1,19-bilindione can adopt a number of conformations since it has exocyclic three double bonds and three single bonds that are rotatable thermally and photochemically. In solution, biliverdin and phycocyanobilin adopt a cyclic helical ZZZ, syn, syn, syn conformation, but other conformations are stabilized depending on the experimental conditions and substituents on the bilin framework. The conformational changes in 1,19-bilindiones are related to the biological functions of a photoreceptor protein, phytochrome. Structural and conformational studies of bilindiones are summarized both in solution and in protein. The conformational changes of bilins can be used for other functions such as a chirality sensor. The bilindiones and the zinc complexes of bilindiones can be employed as a chirality sensor due to the helically chiral structure and the dynamics of racemization of enantiomers. In this paper, we discuss the conformational equilibria and dynamics of bilindiones and its implications in photobiology and materials science.
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Li, Haiyan, Zanxia Cao, Guodong Hu, Liling Zhao, Chunling Wang, and Jihua Wang. "Ligand-induced structural changes analysis of ribose-binding protein as studied by molecular dynamics simulations." Technology and Health Care 29 (March 25, 2021): 103–14. http://dx.doi.org/10.3233/thc-218011.
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BACKGROUND: The ribose-binding protein (RBP) from Escherichia coli is one of the representative structures of periplasmic binding proteins. Binding of ribose at the cleft between two domains causes a conformational change corresponding to a closure of two domains around the ligand. The RBP has been crystallized in the open and closed conformations. OBJECTIVE: With the complex trajectory as a control, our goal was to study the conformation changes induced by the detachment of the ligand, and the results have been revealed from two computational tools, MD simulations and elastic network models. METHODS: Molecular dynamics (MD) simulations were performed to study the conformation changes of RBP starting from the open-apo, closed-holo and closed-apo conformations. RESULTS: The evolution of the domain opening angle θ clearly indicates large structural changes. The simulations indicate that the closed states in the absence of ribose are inclined to transition to the open states and that ribose-free RBP exists in a wide range of conformations. The first three dominant principal motions derived from the closed-apo trajectories, consisting of rotating, bending and twisting motions, account for the major rearrangement of the domains from the closed to the open conformation. CONCLUSIONS: The motions showed a strong one-to-one correspondence with the slowest modes from our previous study of RBP with the anisotropic network model (ANM). The results obtained for RBP contribute to the generalization of robustness for protein domain motion studies using either the ANM or PCA for trajectories obtained from MD.
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Fernández-Quintero, Monica L., Martin C. Heiss, and Klaus R. Liedl. "Antibody humanization—the Influence of the antibody framework on the CDR-H3 loop ensemble in solution." Protein Engineering, Design and Selection 32, no. 9 (2019): 411–22. http://dx.doi.org/10.1093/protein/gzaa004.
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Abstract Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the complementarity determining region (CDR)-H3 loop dynamics. We also studied a Fv without structural information of the anti-idiotypic antibody Ab2/3H6, because it completely lost its binding affinity upon superhumanization, as an example of a failed humanization. Enhanced sampling techniques in combination with molecular dynamics simulations allow to access micro- to milli-second timescales of the CDR-H3 loop dynamics and reveal kinetic and thermodynamic changes involved in the process of humanization. In most cases, we observe a reduced conformational diversity of the CDR-H3 loop when grafted on a human framework and find a conformational shift of the dominant CDR-H3 loop conformation in solution. A shallow side minimum of the conformational CDR-H3 loop ensemble attached to the murine framework becomes the dominant conformation in solution influenced by the human framework. Additionally, we observe in the case of the failed humanization that the potentially binding competent murine CDR-H3 loop ensemble in solution shows nearly no kinetical or structural overlap with the superhumanized variant, thus explaining the loss of binding.
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Ohhashi, Yumiko, Yoshiki Yamaguchi, Hiroshi Kurahashi, et al. "Molecular basis for diversification of yeast prion strain conformation." Proceedings of the National Academy of Sciences 115, no. 10 (2018): 2389–94. http://dx.doi.org/10.1073/pnas.1715483115.
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Self-propagating β-sheet–rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.
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Lenaz, Giorgio. "Lipid fluidity and membrane protein dynamics." Bioscience Reports 7, no. 11 (1987): 823–37. http://dx.doi.org/10.1007/bf01119473.
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Membrane fluidity plays an important role in cellular functions. Membrane proteins are mobile in the lipid fluid environment; lateral diffusion of membrane proteins is slower than expected by theory, due to both the effect of protein crowding in the membrane and to constraints from the aqueous matrix. A major aspect of diffusion is in macromolecular associations: reduction of dimensionality for membrane diffusion facilitates collisional encounters, as those concerned with receptor-mediated signal transduction and with electron transfer chains. In mitochondrial electron transfer, diffusional control is prevented by the excess of collisional encounters between fast-diffusing ubiquinone and the respiratory complexes. Another aspect of dynamics of membrane proteins is their conformational flexibility. Lipids may induce the optimal conformation for catalytic activity. Breaks in Arrhenius plots of membrane-bound enzymes may be related to lipid fluidity: the break could occur when a limiting viscosity is reached for catalytic activity. Viscosity can affect protein conformational changes by inhibiting thermal fluctuations to the inner core of the protein molecule.
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Ludwiczak, Jan, Ewa Szczęsna, Antônio Marinho da Silva Neto, Piotr Cieplak, Andrzej A. Kasprzak, and Adam Jarmuła. "Interactions between motor domains in kinesin-14 Ncd — a molecular dynamics study." Biochemical Journal 476, no. 17 (2019): 2449–62. http://dx.doi.org/10.1042/bcj20190484.
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Abstract Minus-end directed, non-processive kinesin-14 Ncd is a dimeric protein with C-terminally located motor domains (heads). Generation of the power-stroke by Ncd consists of a lever-like rotation of a long superhelical ‘stalk’ segment while one of the kinesin's heads is bound to the microtubule. The last ∼30 amino acids of Ncd head play a crucial but still poorly understood role in this process. Here, we used accelerated molecular dynamics simulations to explore the conformational dynamics of several systems built upon two crystal structures of Ncd, the asymmetrical T436S mutant in pre-stroke/post-stroke conformations of two partner subunits and the symmetrical wild-type protein in pre-stroke conformation of both subunits. The results revealed a new conformational state forming following the inward motion of the subunits and stabilized with several hydrogen bonds to residues located on the border or within the C-terminal linker, i.e. a modeled extension of the C-terminus by residues 675–683. Forming of this new, compact Ncd conformation critically depends on the length of the C-terminus extending to at least residue 681. Moreover, the associative motion leading to the compact conformation is accompanied by a partial lateral rotation of the stalk. We propose that the stable compact conformation of Ncd may represent an initial state of the working stroke.
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Caldararu, Octav, Vilhelm Ekberg, Derek T. Logan, Esko Oksanen, and Ulf Ryde. "Exploring ligand dynamics in protein crystal structures with ensemble refinement." Acta Crystallographica Section D Structural Biology 77, no. 8 (2021): 1099–115. http://dx.doi.org/10.1107/s2059798321006513.
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Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein–ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein–ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and R free values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.
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Ramirez-Mondragon, Carlos A., Megin E. Nguyen, Jozafina Milicaj, et al. "Conserved Conformational Hierarchy across Functionally Divergent Glycosyltransferases of the GT-B Structural Superfamily as Determined from Microsecond Molecular Dynamics." International Journal of Molecular Sciences 22, no. 9 (2021): 4619. http://dx.doi.org/10.3390/ijms22094619.
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It has long been understood that some proteins undergo conformational transitions en route to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site triggers an open-to-closed conformation transition, necessary for their catalytic functions. Herein, we describe microsecond molecular dynamics simulations of two distantly related glycosyltransferases that are part of the GT-B structural superfamily, HepI and GtfA. Simulations were performed using the open and closed conformations of these unbound proteins, respectively, and we sought to identify the major dynamical modes and communication networks that interconnect the open and closed structures. We provide the first reported evidence within the scope of our simulation parameters that the interconversion between open and closed conformations is a hierarchical multistep process which can be a conserved feature of enzymes of the same structural superfamily. Each of these motions involves of a collection of smaller molecular reorientations distributed across both domains, highlighting the complexities of protein dynamic involved in the interconversion process. Additionally, dynamic cross-correlation analysis was employed to explore the potential effect of distal residues on the catalytic efficiency of HepI. Multiple distal nonionizable residues of the C-terminal domain exhibit motions anticorrelated to positively charged residues in the active site in the N-terminal domain involved in substrate binding. Mutations of these residues resulted in a reduction in negatively correlated motions and an altered enzymatic efficiency that is dominated by lower Km values with kcat effectively unchanged. The findings suggest that residues with opposing conformational motions involved in the opening and closing of the bidomain HepI protein can allosterically alter the population and conformation of the “closed” state, essential to the formation of the Michaelis complex. The stabilization effects of these mutations likely equally influence the energetics of both the ground state and the transition state of the catalytic reaction, leading to the unaltered kcat. Our study provides new insights into the role of conformational dynamics in glycosyltransferase’s function and new modality to modulate enzymatic efficiency.
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Yun, R. H., and Jan Hermans. "Conformation equilibria of valine studies by dynamics simulation." "Protein Engineering, Design and Selection" 4, no. 7 (1991): 761–66. http://dx.doi.org/10.1093/protein/4.7.761.
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Guo, Qing, Yufan He, and H. Peter Lu. "Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy." Proceedings of the National Academy of Sciences 112, no. 45 (2015): 13904–9. http://dx.doi.org/10.1073/pnas.1506405112.
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Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions.
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Campbell, Ashley C., Kyle M. Stiers, Julia S. Martin Del Campo, Ritcha Mehra-Chaudhary, Pablo Sobrado, and John J. Tanner. "Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics." Journal of Biological Chemistry 295, no. 38 (2020): 13239–49. http://dx.doi.org/10.1074/jbc.ra120.014750.
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The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase from Aspergillus fumigatus is a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is an N-monooxygenase that catalyzes the NADPH-dependent hydroxylation of l-ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) or l-ornithine bound (resting state). The resting state structure reveals a new out active site conformation characterized by large rotations of the FAD isoalloxazine around the C1–′C2′ and N10–C1′ bonds, coupled to a 10-Å movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to the in conformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) and l-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of the out conformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.
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Roh, Soung-Hun, Corey F. Hryc, Hyun-Hwan Jeong, et al. "Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM." Proceedings of the National Academy of Sciences 114, no. 31 (2017): 8259–64. http://dx.doi.org/10.1073/pnas.1704725114.
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Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10–12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.
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Higo, J., and H. Umeyama. "Protein dynamics determined by backbone conformation and atom packing." Protein Engineering Design and Selection 10, no. 4 (1997): 373–80. http://dx.doi.org/10.1093/protein/10.4.373.
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Day, Austin L., Per Greisen, Lindsey Doyle, et al. "Unintended specificity of an engineered ligand-binding protein facilitated by unpredicted plasticity of the protein fold." Protein Engineering, Design and Selection 31, no. 10 (2018): 375–87. http://dx.doi.org/10.1093/protein/gzy031.
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Abstract Attempts to create novel ligand-binding proteins often focus on formation of a binding pocket with shape complementarity against the desired ligand (particularly for compounds that lack distinct polar moieties). Although designed proteins often exhibit binding of the desired ligand, in some cases they display unintended recognition behavior. One such designed protein, that was originally intended to bind tetrahydrocannabinol (THC), was found instead to display binding of 25-hydroxy-cholecalciferol (25-D3) and was subjected to biochemical characterization, further selections for enhanced 25-D3 binding affinity and crystallographic analyses. The deviation in specificity is due in part to unexpected altertion of its conformation, corresponding to a significant change of the orientation of an α-helix and an equally large movement of a loop, both of which flank the designed ligand-binding pocket. Those changes led to engineered protein constructs that exhibit significantly more contacts and complementarity towards the 25-D3 ligand than the initial designed protein had been predicted to form towards its intended THC ligand. Molecular dynamics simulations imply that the initial computationally designed mutations may contribute to the movement of the helix. These analyses collectively indicate that accurate prediction and control of backbone dynamics conformation, through a combination of improved conformational sampling and/or de novo structure design, represents a key area of further development for the design and optimization of engineered ligand-binding proteins.
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Laugwitz, Jeannette M., Haleh H. Haeri, Anette Kaiser, et al. "Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements." Molecules 25, no. 18 (2020): 4143. http://dx.doi.org/10.3390/molecules25184143.
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The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.
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Nehls, Thomas, Tim Heymann, Christian Meyners, Felix Hausch, and Frederik Lermyte. "Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation." International Journal of Molecular Sciences 22, no. 18 (2021): 9927. http://dx.doi.org/10.3390/ijms22189927.
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In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the ‘four-dimensional’ protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the ‘toolbox’ of mass spectrometry researchers studying higher-order protein structure.
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Mary, Sophie, Jean-Alain Fehrentz, Marjorie Damian, et al. "How ligands and signalling proteins affect G-protein-coupled receptors' conformational landscape." Biochemical Society Transactions 41, no. 1 (2013): 144–47. http://dx.doi.org/10.1042/bst20120267.
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The dynamic character of GPCRs (G-protein-coupled receptors) is essential to their function. However, the details of how ligands and signalling proteins stabilize a receptor conformation to trigger the activation of a given signalling pathway remain largely unexplored. Multiple data, including recent results obtained with the purified ghrelin receptor, suggest a model where ligand efficacy and functional selectivity are directly related to different receptor conformations. Importantly, distinct effector proteins (G-proteins and arrestins) as well as ligands are likely to affect the conformational landscape of GPCRs in different manners, as we show with the isolated ghrelin receptor. Such modulation of the GPCR conformational landscape by pharmacologically distinct ligands and effector proteins has major implications for the design of new drugs that activate specific signalling pathways.
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Augestad, Elias H., Matteo Castelli, Nicola Clementi, et al. "Global and local envelope protein dynamics of hepatitis C virus determine broad antibody sensitivity." Science Advances 6, no. 35 (2020): eabb5938. http://dx.doi.org/10.1126/sciadv.abb5938.
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Broad antibody sensitivity differences of hepatitis C virus (HCV) isolates and their ability to persist in the presence of neutralizing antibodies (NAbs) remain poorly understood. Here, we show that polymorphisms within glycoprotein E2, including hypervariable region 1 (HVR1) and antigenic site 412 (AS412), broadly affect NAb sensitivity by shifting global envelope protein conformation dynamics between theoretical “closed,” neutralization-resistant and “open,” neutralization-sensitive states. The conformational space of AS412 was skewed toward β-hairpin–like conformations in closed states, which also depended on HVR1, assigning function to these enigmatic E2 regions. Scavenger receptor class B, type I entry dependency of HCV was associated with NAb resistance and correlated perfectly with decreased virus propensity to interact with HCV co-receptor CD81, indicating that decreased NAb sensitivity resulted in a more complex entry pathway. This link between global E1/E2 states and functionally distinct AS412 conformations has important implications for targeting AS412 in rational HCV vaccine designs.
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Giampà, Marco, and Elvira Sgobba. "Insight to Functional Conformation and Noncovalent Interactions of Protein-Protein Assembly Using MALDI Mass Spectrometry." Molecules 25, no. 21 (2020): 4979. http://dx.doi.org/10.3390/molecules25214979.
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Noncovalent interactions are the keys to the structural organization of biomolecule e.g., proteins, glycans, lipids in the process of molecular recognition processes e.g., enzyme-substrate, antigen-antibody. Protein interactions lead to conformational changes, which dictate the functionality of that protein-protein complex. Besides biophysics techniques, noncovalent interaction and conformational dynamics, can be studied via mass spectrometry (MS), which represents a powerful tool, due to its low sample consumption, high sensitivity, and label-free sample. In this review, the focus will be placed on Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) and its role in the analysis of protein-protein noncovalent assemblies exploring the relationship within noncovalent interaction, conformation, and biological function.
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Goricanec, David, Ralf Stehle, Pascal Egloff та ін. "Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding". Proceedings of the National Academy of Sciences 113, № 26 (2016): E3629—E3638. http://dx.doi.org/10.1073/pnas.1604125113.
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Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein–coupled receptor (GPCR) activation. Agonist–receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.
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Gormal, Rachel S., Pranesh Padmanabhan, Ravikiran Kasula та ін. "Modular transient nanoclustering of activated β2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies". Proceedings of the National Academy of Sciences 117, № 48 (2020): 30476–87. http://dx.doi.org/10.1073/pnas.2007443117.
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None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β2-adrenoreceptor (β2-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β2-AR(Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β2-AR(Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.
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Moore, Alexander F., David J. Newman, Shoba Ranganathan, and Fei Liu. "Imaginative Order from Reasonable Chaos: Conformation-Driven Activity and Reactivity in Exploring Protein–Ligand Interactions." Australian Journal of Chemistry 71, no. 12 (2018): 917. http://dx.doi.org/10.1071/ch18416.
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Sir Derek Barton’s seminal work on steroid conformational analysis opened up a new era of enquiry into how the preferred conformation of any molecule could have profound effects on its physical–chemical properties and activities. Conformation-based effects on molecular activity and reactivity continue to manifest, with one key area of investigation currently focussed on conformational entropy in driving protein–ligand interactions. Carrying on from Barton’s initial insight on natural product conformational properties, new questions now address how conformational flexibility within a bioactive natural product structural framework (reasonable chaos), can be directed to confer dynamically new protein–ligand interactions beyond the basic lock–key model (imaginative order). Here we summarise our work on exploring conformational diversity from fluorinated natural product fragments, and how this approach of conformation-coupled diversity-oriented synthesis can be used to iteratively derive ligands with enhanced specificity against highly homologous protein domains. Our results demonstrate that the conformation entropic states of highly conserved protein domains differ significantly, and this conformational diversity, beyond primary sequence analysis, can be duly captured and exploited by natural-product derived ligands with complementary conformational dynamics for enhancing recognition specificity in drug lead discovery.
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Cao, Thong M., and Michael R. King. "Stabilization of the Hinge Region of Human E-selectin Enhances Binding Affinity to Ligands Under Force." Cellular and Molecular Bioengineering 14, no. 1 (2021): 65–74. http://dx.doi.org/10.1007/s12195-021-00666-z.
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Abstract Introduction E-selectin is a member of the selectin family of cell adhesion molecules expressed on the plasma membrane of inflamed endothelium and facilitates initial leukocyte tethering and subsequent cell rolling during the early stages of the inflammatory response via binding to glycoproteins expressing sialyl LewisX and sialyl LewisA (sLeX/A). Existing crystal structures of the extracellular lectin/EGF-like domain of E-selectin complexed with sLeX have revealed that E-selectin can exist in two conformation states, a low affinity (bent) conformation, and a high affinity (extended) conformation. The differentiating characteristic of the two conformations is the interdomain angle between the lectin and the EGF-like domain. Methods Using molecular dynamics (MD) simulations we observed that in the absence of tensile force E-selectin undergoes spontaneous switching between the two conformational states at equilibrium. A single amino acid substitution at residue 2 (serine to tyrosine) on the lectin domain favors the extended conformation. Results Steered molecular dynamics (SMD) simulations of E-selectin and PSGL-1 in conjunction with experimental cell adhesion assays show a longer binding lifetime of E-selectin (S2Y) to PSGL-1 compared to wildtype protein. Conclusions The findings in this study advance our understanding into how the structural makeup of E-selectin allosterically influences its adhesive dynamics.
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Dobrovolska, Olena, Øyvind Strømland, Ørjan Sele Handegård, et al. "Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles." Molecules 26, no. 12 (2021): 3607. http://dx.doi.org/10.3390/molecules26123607.
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The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.
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Martini, Silvia, Claudia Bonechi, Alberto Foletti, and Claudio Rossi. "Water-Protein Interactions: The Secret of Protein Dynamics." Scientific World Journal 2013 (2013): 1–6. http://dx.doi.org/10.1155/2013/138916.
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Water-protein interactions help to maintain flexible conformation conditions which are required for multifunctional protein recognition processes. The intimate relationship between the protein surface and hydration water can be analyzed by studying experimental water properties measured in protein systems in solution. In particular, proteins in solution modify the structure and the dynamics of the bulk water at the solute-solvent interface. The ordering effects of proteins on hydration water are extended for several angstroms. In this paper we propose a method for analyzing the dynamical properties of the water molecules present in the hydration shells of proteins. The approach is based on the analysis of the effects of protein-solvent interactions on water protons NMR relaxation parameters. NMR relaxation parameters, especially the nonselective (R1NS) and selective (R1SE) spin-lattice relaxation rates of water protons, are useful for investigating the solvent dynamics at the macromolecule-solvent interfaces as well as the perturbation effects caused by the water-macromolecule interactions on the solvent dynamical properties. In this paper we demonstrate that Nuclear Magnetic Resonance Spectroscopy can be used to determine the dynamical contributions of proteins to the water molecules belonging to their hydration shells.
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Camacho, Inês S., Alina Theisen, Linus O. Johannissen, et al. "Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor." Proceedings of the National Academy of Sciences 116, no. 4 (2019): 1116–25. http://dx.doi.org/10.1073/pnas.1813254116.
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UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light, resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C terminus is required for functional binding to signaling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photoactivation. We show that, while truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exists in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C terminus is primed for activation. In the monomer the extended C terminus destabilizes the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.
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Westenhoff, Sebastian, Elena Nazarenko, Erik Malmerberg, Jan Davidsson, Gergely Katona, and Richard Neutze. "Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches." Acta Crystallographica Section A Foundations of Crystallography 66, no. 2 (2010): 207–19. http://dx.doi.org/10.1107/s0108767309054361.
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Proteins undergo conformational changes during their biological function. As such, a high-resolution structure of a protein's resting conformation provides a starting point for elucidating its reaction mechanism, but provides no direct information concerning the protein's conformational dynamics. Several X-ray methods have been developed to elucidate those conformational changes that occur during a protein's reaction, including time-resolved Laue diffraction and intermediate trapping studies on three-dimensional protein crystals, and time-resolved wide-angle X-ray scattering and X-ray absorption studies on proteins in the solution phase. This review emphasizes the scope and limitations of these complementary experimental approaches when seeking to understand protein conformational dynamics. These methods are illustrated using a limited set of examples including myoglobin and haemoglobin in complex with carbon monoxide, the simple light-driven proton pump bacteriorhodopsin, and the superoxide scavenger superoxide reductase. In conclusion, likely future developments of these methods at synchrotron X-ray sources and the potential impact of emerging X-ray free-electron laser facilities are speculated upon.
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Kim, J. I., K. Eom, and S. Na. "Mechanical Mass-Spring Model for Understanding Globular Motion of Proteins." Journal of Mechanics 32, no. 2 (2016): 123–29. http://dx.doi.org/10.1017/jmech.2015.109.
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AbstractThe conformational (structural) change of proteins plays an essential role in their functions. Experiments have been conducted to try to understand the conformational change of proteins, but they have not been successful in providing information on the atomic scale. Simulation methods have been developed to understand the conformational change at an atomic scale in detail. Coarse-grained methods have been developed to calculate protein dynamics with computational efficiency when compared with than all-atom models. A structure-based mass-spring model called the elastic network model (ENM) showed excellent performance in various protein studies. Coarse-grained ENM was modified in various ways to improve the computational efficiency, and consequently to reduce required computational cost for studying the large-scale protein structures. Our previous studies report a modified mass-spring model, which was developed based on condensation method applicable to ENM, and show that the model is able to accurately predict the fluctuation behavior of proteins. We applied this modified mass-spring model to analyze the conformational changes in proteins. We consider two model proteins as an example, where these two proteins exhibit different functions and molecular sizes. It is shown that the modified mass-spring model allows for accurately predicting the pathways of conformation changes for proteins. Our model provides structural insights into the conformation change of proteins related to the biological functions of large protein complexes.
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Conrad, Marcus, Christian A. Söldner, Yinglong Miao, and Heinrich Sticht. "Agonist Binding and G Protein Coupling in Histamine H2 Receptor: A Molecular Dynamics Study." International Journal of Molecular Sciences 21, no. 18 (2020): 6693. http://dx.doi.org/10.3390/ijms21186693.
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The histamine H2 receptor (H2R) plays an important role in the regulation of gastric acid secretion. Therefore, it is a main drug target for the treatment of gastroesophageal reflux or peptic ulcer disease. However, there is as of yet no 3D-structural information available hampering a mechanistic understanding of H2R. Therefore, we created a model of the histamine-H2R-Gs complex based on the structure of the ternary complex of the β2-adrenoceptor and investigated the conformational stability of this active GPCR conformation. Since the physiologically relevant motions with respect to ligand binding and conformational changes of GPCRs can only partly be assessed on the timescale of conventional MD (cMD) simulations, we also applied metadynamics and Gaussian accelerated molecular dynamics (GaMD) simulations. A multiple walker metadynamics simulation in combination with cMD was applied for the determination of the histamine binding mode. The preferential binding pose detected is in good agreement with previous data from site directed mutagenesis and provides a basis for rational ligand design. Inspection of the H2R-Gs interface reveals a network of polar interactions that may contribute to H2R coupling selectivity. The cMD and GaMD simulations demonstrate that the active conformation is retained on a μs-timescale in the ternary histamine-H2R-Gs complex and in a truncated complex that contains only Gs helix α5 instead of the entire G protein. In contrast, histamine alone is unable to stabilize the active conformation, which is in line with previous studies of other GPCRs.
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Aljoundi, Aimen, Ahmed El Rashedy, Patrick Appiah-Kubi та Mahmoud E. S. Soliman. "Coupling of HSP72 α-Helix Subdomains by the Unexpected Irreversible Targeting of Lysine-56 over Cysteine-17; Coevolution of Covalent Bonding". Molecules 25, № 18 (2020): 4239. http://dx.doi.org/10.3390/molecules25184239.
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Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The unexpected covalent inhibition of heat shock protein 72 (HSP72) by covalently targeting Lys-56 instead of Cys-17 was an interesting observation. However, the structural basis and conformational changes associated with this preferential coupling to Lys-56 over Cys-17 remain unclear. To resolve this mystery, we employed structural and dynamic analyses to investigate the structural basis and conformational dynamics associated with the unexpected covalent inhibition. Our analyses reveal that the coupling of the irreversible inhibitor to Lys-56 is intrinsically less dynamic than Cys-17. Conformational dynamics analyses further reveal that the coupling of the inhibitor to Lys-56 induced a closed conformation of the nucleotide-binding subdomain (NBD) α-helices, in contrast, an open conformation was observed in the case of Cys-17. The closed conformation maintained the crucial salt-bridge between Glu-268 and Lys-56 residues, which strengthens the interaction affinity of the inhibitor nearly identical to adenosine triphosphate (ADP/Pi) bound to the HSP72-NBD. The outcome of this report provides a substantial shift in the conventional direction for the design of more potent covalent inhibitors.
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Tyagi, Vivek, Victor Vasquez-Montes, J. Alfredo Freites, Alexander Kyrychenko, Douglas J. Tobias, and Alexey S. Ladokhin. "Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL." International Journal of Molecular Sciences 22, no. 17 (2021): 9388. http://dx.doi.org/10.3390/ijms22179388.
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The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.
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Zabik, Nicole L., Matthew M. Imhof, and Sanela Martic-Milne. "Structural evaluations of tau protein conformation: methodologies and approaches." Biochemistry and Cell Biology 95, no. 3 (2017): 338–49. http://dx.doi.org/10.1139/bcb-2016-0227.
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Protein-misfolding diseases are based on a common principle of aggregation initiated by intra- and inter-molecular contacts. The structural and conformational changes induced by biochemical transformations such as post-translational modifications (PTMs), often lead to protein unfolding and misfolding. Thus, these order-to-disorder or disorder-to-order transitions may regulate cellular function. Tau, a neuronal protein, regulates microtubule (MT) structure and overall cellular integrity. However, misfolded tau modified by PTMs results in MT destabilization, toxic tau aggregate formation, and ultimately cell death, leading to neurodegeneration. Currently, the lack of structural information surrounding tau severely limits understanding of neurodegeneration. This minireview focuses on the current methodologies and approaches aimed at probing tau conformation and the role of conformation in various aspects of tau biochemistry. The recent applications of nuclear magnetic resonance, mass spectrometry, Förster resonance electron transfer, and molecular dynamics simulations toward structural analysis of conformational landscapes of tau will be described. The strategies developed for structural evaluation of tau may significantly improve our understanding of misfolding diseases.
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Damian, Marjorie, Sophie Mary, Mathieu Maingot, et al. "Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex." Proceedings of the National Academy of Sciences 112, no. 5 (2015): 1601–6. http://dx.doi.org/10.1073/pnas.1414618112.
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How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.
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Li, Qingxin, and CongBao Kang. "Insights into Structures and Dynamics of Flavivirus Proteases from NMR Studies." International Journal of Molecular Sciences 21, no. 7 (2020): 2527. http://dx.doi.org/10.3390/ijms21072527.
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Nuclear magnetic resonance (NMR) spectroscopy plays important roles in structural biology and drug discovery, as it is a powerful tool to understand protein structures, dynamics, and ligand binding under physiological conditions. The protease of flaviviruses is an attractive target for developing antivirals because it is essential for the maturation of viral proteins. High-resolution structures of the proteases in the absence and presence of ligands/inhibitors were determined using X-ray crystallography, providing structural information for rational drug design. Structural studies suggest that proteases from Dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV) exist in open and closed conformations. Solution NMR studies showed that the closed conformation is predominant in solution and should be utilized in structure-based drug design. Here, we reviewed solution NMR studies of the proteases from these viruses. The accumulated studies demonstrated that NMR spectroscopy provides additional information to understand conformational changes of these proteases in the absence and presence of substrates/inhibitors. In addition, NMR spectroscopy can be used for identifying fragment hits that can be further developed into potent protease inhibitors.
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Wu, Si, Liu Hong, Yuqing Wang, et al. "Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function." Proceedings of the National Academy of Sciences 117, no. 14 (2020): 7814–23. http://dx.doi.org/10.1073/pnas.1914376117.
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Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.
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Sun, Jixue, Zibin Li, and Na Yang. "Mechanism of the Conformational Change of the Protein Methyltransferase SMYD3: A Molecular Dynamics Simulation Study." International Journal of Molecular Sciences 22, no. 13 (2021): 7185. http://dx.doi.org/10.3390/ijms22137185.
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SMYD3 is a SET-domain-containing methyltransferase that catalyzes the transfer of methyl groups onto lysine residues of substrate proteins. Methylation of MAP3K2 by SMYD3 has been implicated in Ras-driven tumorigenesis, which makes SMYD3 a potential target for cancer therapy. Of all SMYD family proteins, SMYD3 adopt a closed conformation in a crystal structure. Several studies have suggested that the conformational changes between the open and closed forms may regulate the catalytic activity of SMYD3. In this work, we carried out extensive molecular dynamics simulations on a series of complexes with a total of 21 μs sampling to investigate the conformational changes of SMYD3 and unveil the molecular mechanisms. Based on the C-terminal domain movements, the simulated models could be depicted in three different conformational states: the closed, intermediate and open states. Only in the case that both the methyl donor binding pocket and the target lysine-binding channel had bound species did the simulations show SMYD3 maintaining its conformation in the closed state, indicative of a synergetic effect of the cofactors and target lysine on regulating the conformational change of SMYD3. In addition, we performed analyses in terms of structure and energy to shed light on how the two regions might regulate the C-terminal domain movement. This mechanistic study provided insights into the relationship between the conformational change and the methyltransferase activity of SMYD3. The more complete understanding of the conformational dynamics developed here together with further work may lay a foundation for the rational drug design of SMYD3 inhibitors.
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Sharma, Meenakshi, Nancy Jaiswal, Dinesh Kumar, and Krishna Mohan Poluri. "Enhanced dynamics of conformationally heterogeneous T7 bacteriophage lysozyme native state attenuates its stability and activity." Biochemical Journal 476, no. 3 (2019): 613–28. http://dx.doi.org/10.1042/bcj20180703.
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Abstract Proteins are dynamic in nature and exist in a set of equilibrium conformations on various timescale motions. The flexibility of proteins governs various biological functions, and therefore elucidation of such functional dynamics is essential. In this context, we have studied the structure–dynamics–stability–activity relationship of bacteriophage T7 lysozyme/endolysin (T7L) native-state ensemble in the pH range of 6–8. Our studies established that T7L native state is conformationally heterogeneous, as several residues of its C-terminal half are present in two conformations (major and minor) in the slow exchange time scale of nuclear magnetic resonance (NMR). Structural and dynamic studies suggested that the residues belonging to minor conformations do exhibit native-like structural and dynamic features. Furthermore, the NMR relaxation experiments unraveled that the native state is highly dynamic and the dynamic behavior is regulated by the pH, as the pH 6 conformation exhibited enhanced dynamics compared with pH 7 and 8. The stability measurements and cell-based activity studies on T7L indicated that the native protein at pH 6 is ∼2 kcal less stable and is ∼50% less active than those of pH 7 and 8. A comprehensive analysis of the T7L active site, unfolding initiation sites and the residues with altered dynamics outlined that the attenuation of stability and activity is a resultant of its enhanced dynamic properties, which, in turn, can be attributed to the protonation/deprotonation of its partially buried His residues. Our study on T7L structure–dynamics–activity paradigm could assist in engineering novel amidase-based endolysins with enhanced activity and stability over a broad pH range.
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Schafer, Christopher T., Jonathan F. Fay, Jay M. Janz, and David L. Farrens. "Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state." Proceedings of the National Academy of Sciences 113, no. 42 (2016): 11961–66. http://dx.doi.org/10.1073/pnas.1606347113.
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Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.
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Lee, Ho-Sup, Yulia A. Komarova, Elena S. Nadezhdina, et al. "Phosphorylation Controls Autoinhibition of Cytoplasmic Linker Protein-170." Molecular Biology of the Cell 21, no. 15 (2010): 2661–73. http://dx.doi.org/10.1091/mbc.e09-12-1036.
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Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an “open” conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the “folded back” conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.
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Langan, Patricia S., Venu Gopal Vandavasi, Wojciech Kopec, et al. "The structure of a potassium-selective ion channel reveals a hydrophobic gate regulating ion permeation." IUCrJ 7, no. 5 (2020): 835–43. http://dx.doi.org/10.1107/s2052252520008271.
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Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydrophobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydrophobic gate, regulating the flow of ions through the selectivity filter.
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JANUAR, M., A. SULAIMAN, and L. T. HANDOKO. "NONLINEAR CONFORMATION OF SECONDARY PROTEIN FOLDING." International Journal of Modern Physics: Conference Series 09 (January 2012): 127–32. http://dx.doi.org/10.1142/s2010194512005181.
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A model to describe the mechanism of conformational dynamics in secondary protein based on matter interactions is proposed. The approach deploys the lagrangian method by imposing certain symmetry breaking. The protein backbone is initially assumed to be nonlinear and represented by the Sine-Gordon equation, while the nonlinear external bosonic sources is represented by ϕ4 interaction. It is argued that the nonlinear source induces the folding pathway in a different way than the previous work with initially linear backbone. Also, the nonlinearity of protein backbone decreases the folding speed.
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Moorthy, Balakrishnan, Lavanya Iyer, and Elizabeth Topp. "Characterizing Protein Structure, Dynamics and Conformation in Lyophilized Solids." Current Pharmaceutical Design 21, no. 40 (2015): 5845–53. http://dx.doi.org/10.2174/1381612821666151008150735.
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Yang, Haw. "Single-Molecule FRET for Protein Conformation Distributions and Dynamics." Biophysical Journal 102, no. 3 (2012): 42a. http://dx.doi.org/10.1016/j.bpj.2011.11.262.
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Goldberg, Mohel E. "Investigating protein conformation dynamics and folding with monoclonal antibodies." Trends in Biochemical Sciences 16 (January 1991): 358–62. http://dx.doi.org/10.1016/0968-0004(91)90148-o.
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Brouhard, Gary J., та Luke M. Rice. "The contribution of αβ-tubulin curvature to microtubule dynamics". Journal of Cell Biology 207, № 3 (2014): 323–34. http://dx.doi.org/10.1083/jcb.201407095.
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Microtubules are dynamic polymers of αβ-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized αβ-tubulin. The ability of αβ-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. αβ-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks.
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Stewart, Chelsea M., Cosmo Z. Buffalo, J. Andrés Valderrama, et al. "Coiled-coil destabilizing residues in the group A Streptococcus M1 protein are required for functional interaction." Proceedings of the National Academy of Sciences 113, no. 34 (2016): 9515–20. http://dx.doi.org/10.1073/pnas.1606160113.
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The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A “capture-and-collapse” model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.
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Papaleo, Elena, Carlo Camilloni, Kaare Teilum, Michele Vendruscolo, and Kresten Lindorff-Larsen. "Molecular dynamics ensemble refinement of the heterogeneous native state of NCBD using chemical shifts and NOEs." PeerJ 6 (July 4, 2018): e5125. http://dx.doi.org/10.7717/peerj.5125.
Full textAbstract:
Many proteins display complex dynamical properties that are often intimately linked to their biological functions. As the native state of a protein is best described as an ensemble of conformations, it is important to be able to generate models of native state ensembles with high accuracy. Due to limitations in sampling efficiency and force field accuracy it is, however, challenging to obtain accurate ensembles of protein conformations by the use of molecular simulations alone. Here we show that dynamic ensemble refinement, which combines an accurate atomistic force field with commonly available nuclear magnetic resonance (NMR) chemical shifts and NOEs, can provide a detailed and accurate description of the conformational ensemble of the native state of a highly dynamic protein. As both NOEs and chemical shifts are averaged on timescales up to milliseconds, the resulting ensembles reflect the structural heterogeneity that goes beyond that probed, e.g., by NMR relaxation order parameters. We selected the small protein domain NCBD as object of our study since this protein, which has been characterized experimentally in substantial detail, displays a rich and complex dynamical behaviour. In particular, the protein has been described as having a molten-globule like structure, but with a relatively rigid core. Our approach allowed us to describe the conformational dynamics of NCBD in solution, and to probe the structural heterogeneity resulting from both short- and long-timescale dynamics by the calculation of order parameters on different time scales. These results illustrate the usefulness of our approach since they show that NCBD is rather rigid on the nanosecond timescale, but interconverts within a broader ensemble on longer timescales, thus enabling the derivation of a coherent set of conclusions from various NMR experiments on this protein, which could otherwise appear in contradiction with each other.
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Devlin, Jason, Jesus Alonso, Grant Keller, Sara Bobisse, Alexandre Harari, and Brian Baker. "4094 Structural Determinants of Immunogenicity for Peptide-Based Immunotherapy." Journal of Clinical and Translational Science 4, s1 (2020): 16. http://dx.doi.org/10.1017/cts.2020.92.
Full textAbstract:
OBJECTIVES/GOALS: Neoantigen vaccine immunotherapies have shown promise in clinical trials, but identifying which peptides to include in a vaccine remains a challenge. We aim to establish that molecular structural features can help predict which neoantigens to target to achieve tumor regression. METHODS/STUDY POPULATION: Proteins were prepared by recombinant expression in E. coli followed by in vitro refolding. Correctly folded proteins were purified by chromatography. Affinities of protein-protein interactions were measured by surface plasmon resonance (SPR) and thermal stabilities of proteins were determined by differential scanning fluorimetry. All experiments were performed at least in triplicate. Protein crystals were obtained by hanging drop vapor diffusion. The protein crystal structures were solved by molecular replacement and underwent several rounds of automated refinement. Molecular dynamics simulations were performed using the AMBER molecular dynamics package. RESULTS/ANTICIPATED RESULTS: A T cell receptor (TCR) expressed by tumor-infiltrating T cells exhibited a 20-fold stronger binding affinity to the neoantigen peptide compared to the self-peptide. X-ray crystal structures of the peptides with the major histocompatibility complex (MHC) protein demonstrated that a non-mutated residue in the peptide samples different positions with the mutation. The difference in conformations of the non-mutated residue was supported by molecular dynamics simulations. Crystal structures of the TCR engaging both peptide/MHCs suggested that the conformation favored by the mutant peptide was crucial for TCR binding. The TCR bound the neoantigen/MHC with faster binding kinetics. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that the mutation impacts the conformation of another residue in the peptide, and this alteration allows for more favorable T cell receptor binding to the neoantigen. This highlights the potential of non-mutated residues in contributing to neoantigen recognition.