RESUMEN
Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.
Asunto(s)
Biotinilación , Esteroles , Proteínas de Unión al GTP rab , Humanos , Colesterol/biosíntesis , Colesterol/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Esteroles/biosíntesis , Esteroles/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Transporte de Proteínas/genéticaRESUMEN
Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.
Asunto(s)
Proteínas Bacterianas , Proteínas de la Membrana , Streptococcus gordonii , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Streptococcus gordonii/química , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismoRESUMEN
Streptococcus groups A and B cause serious infections, including early onset sepsis and meningitis in newborns. Rib domain-containing surface proteins are found associated with invasive strains and elicit protective immunity in animal models. Yet, despite their apparent importance in infection, the structure of the Rib domain was previously unknown. Structures of single Rib domains of differing length reveal a rare case of domain atrophy through deletion of 2 core antiparallel strands, resulting in the loss of an entire sheet of the ß-sandwich from an immunoglobulin-like fold. Previously, observed variation in the number of Rib domains within these bacterial cell wall-attached proteins has been suggested as a mechanism of immune evasion. Here, the structure of tandem domains, combined with molecular dynamics simulations and small angle X-ray scattering, suggests that variability in Rib domain number would result in differential projection of an N-terminal host-colonization domain from the bacterial surface. The identification of 2 further structures where the typical B-D-E immunoglobulin ß-sheet is replaced with an α-helix further confirms the extensive structural malleability of the Rib domain.
RESUMEN
Cytochrome P450cam (CYP101A1) catalyzes the regio- and stereo-specific 5-exo-hydroxylation of camphor via a multistep catalytic cycle that involves two-electron transfer steps, with an absolute requirement that the second electron be donated by the ferrodoxin, putidaredoxin (Pdx). Whether P450cam, once camphor has bound to the active site and the substrate entry channel has closed, opens up upon Pdx binding, during the second electron transfer step, or it remains closed is still a matter of debate. A potential allosteric site for camphor binding has been identified and postulated to play a role in the binding of Pdx. Here, we have revisited paramagnetic NMR spectroscopy data and determined a heterogeneous ensemble of structures that explains the data, provides a complete representation of the P450cam/Pdx complex in solution, and reconciles alternative hypotheses. The allosteric camphor binding site is always present, and the conformational changes induced by camphor binding to this site facilitates Pdx binding. We also determined that the state to which Pdx binds comprises an ensemble of structures that have features of both the open and closed state. These results demonstrate that there is a finely balanced interaction between allosteric camphor binding and the binding of Pdx at high camphor concentrations.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Alcanfor 5-Monooxigenasa/química , Alcanfor 5-Monooxigenasa/metabolismo , Alcanfor/química , Ferredoxinas/metabolismo , Pseudomonas putida/enzimología , Regulación Alostérica , Alcanfor/metabolismo , Dominio Catalítico , Cristalografía por Rayos X/métodos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pseudomonas putida/químicaRESUMEN
Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs.
Asunto(s)
Miosinas/química , Miosinas/metabolismo , Animales , Cristalografía por Rayos X , Ratones , Simulación de Dinámica Molecular , Miosina VIIa , Conformación Proteica en Hélice alfa , Estabilidad ProteicaRESUMEN
Hydrogen/deuterium exchange monitored by mass spectrometry is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is generally not possible. Here, we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) may be extracted and the uniqueness of the solutions obtained depending on the degree of peptide overlap. However, in most cases, the solution is not unique, and multiple alternatives must be considered. We provide a statistical method that clusters the solutions to further reduce their number. Such analysis always provides meaningful constraints on protection factors and can be used in situations in which obtaining more refined experimental data is impractical. It also provides a systematic way to improve data collection strategies to obtain unambiguous information at single-residue level (e.g., for assessing protein structure predictions at atomistic level).
Asunto(s)
Deuterio/química , Espectrometría de Masas/métodos , Péptidos/química , Amidas/química , Complemento C3/química , Enlace de Hidrógeno , Espectrometría de Masas/normasRESUMEN
The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein-protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are prescreened for "designability", limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multisubunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. Analysis of a tubular structure determined by X-ray crystallography has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight "native-state" forced protein-protein interfaces, which may prove useful as starting conformations for future computational design.
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Toxina del Cólera/química , Proteínas/química , Algoritmos , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Dominios ProteicosRESUMEN
Determining how proteins interact to form stable complexes is of crucial importance, for example in the development of novel therapeutics. Computational methods to determine the thermodynamically stable conformation of complexes from the structure of the binding partners, such as RosettaDock, might potentially emerge to become a promising alternative to traditional structure determination methods. However, while models virtually identical to the correct experimental structure can in some cases be generated, the main difficulty remains to discriminate correct or approximately correct models from decoys. This is due to the ruggedness of the free-energy landscape, the approximations intrinsic in the scoring functions, and the intrinsic flexibility of proteins. Here we show that molecular dynamics simulations performed starting from a number top-scoring models can not only discriminate decoys and identify the correct structure, but may also provide information on an initial map of the free energy landscape that elucidates the binding mechanism.
Asunto(s)
Simulación de Dinámica Molecular , Conformación Proteica , Proteínas/química , Proteínas/metabolismo , Biología Computacional , Pliegue de Proteína , Programas Informáticos , TermodinámicaRESUMEN
Many human proteins contain intrinsically disordered regions, and disorder in these proteins can be fundamental to their function-for example, facilitating transient but specific binding, promoting allostery, or allowing efficient posttranslational modification. SasG, a multidomain protein implicated in host colonization and biofilm formation in Staphylococcus aureus, provides another example of how disorder can play an important role. Approximately one-half of the domains in the extracellular repetitive region of SasG are intrinsically unfolded in isolation, but these E domains fold in the context of their neighboring folded G5 domains. We have previously shown that the intrinsic disorder of the E domains mediates long-range cooperativity between nonneighboring G5 domains, allowing SasG to form a long, rod-like, mechanically strong structure. Here, we show that the disorder of the E domains coupled with the remarkable stability of the interdomain interface result in cooperative folding kinetics across long distances. Formation of a small structural nucleus at one end of the molecule results in rapid structure formation over a distance of 10 nm, which is likely to be important for the maintenance of the structural integrity of SasG. Moreover, if this normal folding nucleus is disrupted by mutation, the interdomain interface is sufficiently stable to drive the folding of adjacent E and G5 domains along a parallel folding pathway, thus maintaining cooperative folding.
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Proteínas Intrínsecamente Desordenadas/química , Dominios Proteicos , Pliegue de Proteína , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Intrínsecamente Desordenadas/genética , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , Estabilidad Proteica , Desplegamiento ProteicoRESUMEN
The receptor for glucagon-like peptide 1 (GLP-1R) is a validated drug target for the treatment of type 2 diabetes and obesity. Recently the first three structures of GLP-1R were published - an X-ray structure of the apo transmembrane domain in the inactive conformation; an X-ray structure of the full-length receptor bound to a truncated peptide agonist; and a cryo-EM structure of the full-length receptor bound with GLP-1 and coupled to the G protein Gs. Since the inactive structure was incomplete, and the two active-state structures shared significant differences, we utilised all available knowledge to build hybrid models of the full length active and inactive state receptors. The two models were simulated using molecular dynamics and the output trajectories analysed and compared to reveal insights into the mechanism for agonist-mediated receptor activation. His-7, Glu-9 and Asp-15 of GLP-1 act together to destabilise transmembrane helix 6 and extracellular loop 3 in order to generate an active conformation of GLP-1R.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/química , Simulación de Dinámica Molecular , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
BACKGROUND: The distribution of the length of a polypeptide, or that of the distance between any two of its atoms, is an important property as it can be analytically or numerically estimated for a number of polymer models. Importantly, it is directly measurable through a number of different experimental techniques. Length distributions can be straightforwardly assessed from molecular dynamics simulation; however, true convergence through full accurate coverage of the length range is difficult to achieve. METHODS: The application of external constant force combined with the weighted-histogram analysis method (WHAM) is used to enhance sampling of unlikely 'long' or 'short' conformations and obtain the potential of mean force, while also collecting dynamic properties of the chain under variable tension. RESULTS: We demonstrate the utility of constant force to enhance the sampling efficiency and obtain experimentally measurable quantities on a series of short peptides, including charge-rich sequences that are known to be highly helical but whose properties are distinct from those of helical peptides undergoing helix-coil transitions. CONCLUSIONS: Force-enhanced sampling enhances the range and accuracy of the length-based potential of mean force of the peptide, in particular those sequences that contain increased numbers of charged residues. GENERAL SIGNIFICANCE: This approach allows users to simultaneously probe the force-dependent behaviour of peptides directly, enhance the range and accuracy of the length-based PMF of the peptide and also test the convergence of simulations by comparing the overlap of PMF profiles from different constant forces. This article is part of a special issue entitled Recent developments of molecular dynamics.
Asunto(s)
Simulación de Dinámica Molecular , Oligopéptidos/química , Arginina/química , Ácido Glutámico/química , Glicina/química , Lisina/química , Estructura Secundaria de Proteína , Estrés Mecánico , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
Rotary ATPases are involved in numerous physiological processes, with the three distinct types (F/A/V-ATPases) sharing functional properties and structural features. The basic mechanism involves the counter rotation of two motors, a soluble ATP hydrolyzing/synthesizing domain and a membrane-embedded ion pump connected through a central rotor axle and a stator complex. Within the A/V-ATPase family conformational flexibility of the EG stators has been shown to accommodate catalytic cycling and is considered to be important to function. For the A-ATPase three EG structures have been reported, thought to represent conformational states of the stator during different stages of rotary catalysis. Here we use long, detailed atomistic simulations to show that those structures are conformers explored through thermal fluctuations, but do not represent highly populated states of the EG stator in solution. We show that the coiled coil tail domain has a high persistence length (â¼100 nm), but retains the ability to adapt to different conformational states through the presence of two hinge regions. Moreover, the stator network of the related V-ATPase has been suggested to adapt to subunit interactions in the collar region in addition to the nucleotide occupancy of the catalytic domain. The MD simulations reported here, reinforce this observation showing that the EG stators have enough flexibility to adapt to significantly different structural re-arrangements and accommodate structural changes in the catalytic domain whilst resisting the large torque generated by catalytic cycling. These results are important to understand the role the stators play in the rotary-ATPase mechanism. Proteins 2016; 84:1203-1212. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas/química , ATPasas de Translocación de Protón Mitocondriales/química , Subunidades de Proteína/química , Thermus thermophilus/química , ATPasas de Translocación de Protón Vacuolares/química , Adenosina Trifosfato/química , Biocatálisis , Dominio Catalítico , Simulación de Dinámica Molecular , Análisis de Componente Principal , Estructura Secundaria de Proteína , Rotación , Thermus thermophilus/enzimologíaRESUMEN
Proteins from organisms that have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single-molecule force spectroscopy (SMFS), and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal ß-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (ΔGD-N) of the mutated proteins and show that there is a more significant reduction for TmCSP L40A (ΔΔG = 63%) than TmCSP V62A (ΔΔG = 47%), as might be expected on the basis of the relative reduction in the size of the side chain. By contrast, SMFS measures the mechanical stability (ΔG*) and shows a greater reduction for TmCSP V62A (ΔΔG* = 8.4%) than TmCSP L40A (ΔΔG* = 2.5%). While the impact on the mechanical stability is subtle, the results demonstrate the power of tuning noncovalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provide the opportunity to design proteins with optimized thermodynamic and mechanical properties.
Asunto(s)
Proteínas Bacterianas/química , Termodinámica , Thermotoga maritima/química , Interacciones Hidrofóbicas e Hidrofílicas , Dominios Proteicos , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
The past decade has seen the development of a new class of rare event methods in which molecular configuration space is divided into a set of boundaries/interfaces, and then short trajectories are run between boundaries. For all these methods, an important concern is how to generate boundaries. In this paper, we outline an algorithm for adaptively generating boundaries along a free energy surface in multi-dimensional collective variable (CV) space, building on the boxed molecular dynamics (BXD) rare event algorithm. BXD is a simple technique for accelerating the simulation of rare events and free energy sampling which has proven useful for calculating kinetics and free energy profiles in reactive and non-reactive molecular dynamics (MD) simulations across a range of systems, in both NVT and NVE ensembles. Two key developments outlined in this paper make it possible to automate BXD, and to adaptively map free energy and kinetics in complex systems. First, we have generalized BXD to multidimensional CV space. Using strategies from rigid-body dynamics, we have derived a simple and general velocity-reflection procedure that conserves energy for arbitrary collective variable definitions in multiple dimensions, and show that it is straightforward to apply BXD to sampling in multidimensional CV space so long as the Cartesian gradients ∇CV are available. Second, we have modified BXD to undertake on-the-fly statistical analysis during a trajectory, harnessing the information content latent in the dynamics to automatically determine boundary locations. Such automation not only makes BXD considerably easier to use; it also guarantees optimal boundaries, speeding up convergence. We have tested the multidimensional adaptive BXD procedure by calculating the potential of mean force for a chemical reaction recently investigated using both experimental and computational approaches - i.e., F + CD3CN â DF + D2CN in both the gas phase and a strongly coupled explicit CD3CN solvent. The results obtained using multidimensional adaptive BXD agree well with previously published experimental and computational results, providing good evidence for its reliability.
RESUMEN
Proteins from extremophilic organisms provide excellent model systems to determine the role of non-covalent interactions in defining protein stability and dynamics as well as being attractive targets for the development of robust biomaterials. Hyperthermophilic proteins have a prevalence of salt bridges, relative to their mesophilic homologues, which are thought to be important for enhanced thermal stability. However, the impact of salt bridges on the mechanical properties of proteins is far from understood. Here, a combination of protein engineering, biophysical characterisation, single molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations directly investigates the role of salt bridges in the mechanical stability of two cold shock proteins; BsCSP from the mesophilic organism Bacillus subtilis and TmCSP from the hyperthermophilic organism Thermotoga maritima. Single molecule force spectroscopy shows that at ambient temperatures TmCSP is mechanically stronger yet, counter-intuitively, its native state can withstand greater deformation before unfolding (i.e. it is mechanically soft) compared with BsCSP. MD simulations were used to identify the location and quantify the population of salt bridges, and reveal that TmCSP contains a larger number of highly occupied salt bridges than BsCSP. To test the hypothesis that salt-bridges endow these mechanical properties on the hyperthermophilic CSP, a charged triple mutant (CTM) variant of BsCSP was generated by grafting an ionic cluster from TmCSP into the BsCSP scaffold. As expected CTM is thermodynamically more stable and mechanically softer than BsCSP. We show that a grafted ionic cluster can increase the mechanical softness of a protein and speculate that it could provide a mechanical recovery mechanism and that it may be a design feature applicable to other proteins.
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Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas y Péptidos de Choque por Frío/química , Sales (Química)/química , Thermotoga maritima/química , Secuencia de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Iones/química , Modelos Moleculares , Simulación de Dinámica Molecular , Estabilidad Proteica , Desplegamiento Proteico , Termodinámica , Thermotoga maritima/genéticaRESUMEN
Single α-helix (SAH) domains are rich in charged residues (Arg, Lys, and Glu) and stable in solution over a wide range of pH and salt concentrations. They are found in many different proteins where they bridge two functional domains. To test the idea that their high stability might enable these proteins to resist unfolding along their length, the properties and unfolding behavior of the predicted SAH domain from myosin-10 were characterized. The expressed and purified SAH domain was highly helical, melted non-cooperatively, and was monomeric as shown by circular dichroism and mass spectrometry as expected for a SAH domain. Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (<30 pN) without a characteristic unfolding peak. Molecular dynamics simulations showed that the SAH domain unfolds progressively as the length is increased and refolds progressively as the length is reduced. This enables the SAH domain to act as a constant force spring in the mechanically dynamic environment of the cell.
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Miosinas/química , Animales , Bovinos , Dicroismo Circular , Microscopía de Fuerza Atómica , Modelos Moleculares , Miosinas/genética , Miosinas/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Identifying the contributions to thermodynamic stability of capsids is of fundamental and practical importance. Here we use simulation to assess how mutations affect the stability of lumazine synthase from the hyperthermophile Aquifex aeolicus, a T = 1 icosahedral capsid; in the simulations the icosahedral symmetry of the capsid is preserved by simulating a single pentamer and imposing crystal symmetry, in effect simulating an infinite cubic lattice of icosahedral capsids. The stability is assessed by estimating the free energy of association using an empirical method previously proposed to identify biological units in crystal structures. We investigate the effect on capsid formation of seven mutations, for which it has been experimentally assessed whether they disrupt capsid formation or not. With one exception, our approach predicts the effect of the mutations on the capsid stability. The method allows the identification of interaction networks, which drive capsid assembly, and highlights the plasticity of the interfaces between subunits in the capsid.
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Proteínas Bacterianas/química , Simulación de Dinámica Molecular , Complejos Multienzimáticos/química , Mutación , Conformación Proteica , Bacterias/enzimología , Proteínas Bacterianas/genética , Biología Computacional/métodos , Estabilidad de Enzimas , Interacciones Hidrofóbicas e Hidrofílicas , Complejos Multienzimáticos/genética , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
The human genome contains 39 myosin genes, divided up into 12 different classes. The structure, cellular function and biochemical properties of many of these isoforms remain poorly characterized and there is still some controversy as to whether some myosin isoforms are monomers or dimers. Myosin isoforms 6 and 10 contain a stable single α-helical (SAH) domain, situated just after the canonical lever. The SAH domain is stiff enough to be able to lengthen the lever allowing the myosin to take a larger step. In addition, atomic force microscopy and atomistic simulations show that SAH domains unfold at relatively low forces and have a high propensity to refold. These properties are likely to be important for protein function, enabling motors to carry cargo in dense actin networks, and other proteins to remain attached to binding partners in the crowded cell.
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Miosinas/química , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Miosinas/fisiología , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de ProteínaRESUMEN
The evolution of disease or the progress of recovery of a patient is a complex process, which depends on many factors. A quantitative description of this process in real-time by a single, clinically measurable parameter (biomarker) would be helpful for early, informed and targeted treatment. Organ transplantation is an eminent case in which the evolution of the post-operative clinical condition is highly dependent on the individual case. The quality of management and monitoring of patients after kidney transplant often determines the long-term outcome of the graft. Using NMR spectra of blood samples, taken at different time points from just before to a week after surgery, we have shown that a biomarker can be found that quantitatively monitors the evolution of a clinical condition. We demonstrate that this is possible if the dynamics of the process is considered explicitly: the biomarker is defined and determined as an optimal reaction coordinate that provides a quantitatively accurate description of the stochastic recovery dynamics. The method, originally developed for the analysis of protein folding dynamics, is rigorous, robust and general, i.e., it can be applied in principle to analyze any type of biological dynamics. Such predictive biomarkers will promote improvement of long-term graft survival after renal transplantation, and have potentially unlimited applications as diagnostic tools.