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1.
J Chem Phys ; 155(5): 054102, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34364321

RESUMO

Markov state models (MSMs) have become one of the preferred methods for the analysis and interpretation of molecular dynamics (MD) simulations of conformational transitions in biopolymers. While there is great variation in terms of implementation, a well-defined workflow involving multiple steps is often adopted. Typically, molecular coordinates are first subjected to dimensionality reduction and then clustered into small "microstates," which are subsequently lumped into "macrostates" using the information from the slowest eigenmodes. However, the microstate dynamics is often non-Markovian, and long lag times are required to converge the relevant slow dynamics in the MSM. Here, we propose a variation on this typical workflow, taking advantage of hierarchical density-based clustering. When applied to simulation data, this type of clustering separates high population regions of conformational space from others that are rarely visited. In this way, density-based clustering naturally implements assignment of the data based on transitions between metastable states, resulting in a core-set MSM. As a result, the state definition becomes more consistent with the assumption of Markovianity, and the timescales of the slow dynamics of the system are recovered more effectively. We present results of this simplified workflow for a model potential and MD simulations of the alanine dipeptide and the FiP35 WW domain.


Assuntos
Dipeptídeos/química , Cadeias de Markov , Simulação de Dinâmica Molecular/estatística & dados numéricos , Proteínas/química , Análise por Conglomerados , Conformação Proteica , Domínios WW
2.
ACS Nano ; 15(6): 10203-10216, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34060810

RESUMO

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.


Assuntos
Cardiomiopatia Hipertrófica , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Humanos , Mutação , Fenótipo , Sarcômeros
3.
J Phys Chem B ; 124(41): 8973-8983, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32955882

RESUMO

The rate at which a protein molecule folds is determined by opposing energetic and entropic contributions to the free energy that shape the folding landscape. Delineating the extent to which they impact the diffusional barrier-crossing events, including the magnitude of internal friction and barrier height, has largely been a challenging task. In this work, we extract the underlying thermodynamic and dynamic contributions to the folding rate of an unusually slow-folding helical DNA-binding domain, PurR, which shares the characteristics of ultrafast downhill-folding proteins but nonetheless appears to exhibit an apparent two-state equilibrium. We combine equilibrium spectroscopy, temperature-viscosity-dependent kinetics, statistical mechanical modeling, and coarse-grained simulations to show that the conformational behavior of PurR is highly heterogeneous characterized by a large spread in melting temperatures, marginal thermodynamic barriers, and populated partially structured states. PurR appears to be at the threshold of disorder arising from frustrated electrostatics and weak packing that in turn slows down folding due to a shallow, bumpy landscape and not due to large thermodynamic barriers or strong internal friction. Our work highlights how a strong temperature dependence on the pre-exponential could signal a shallow landscape and not necessarily a slow-folding diffusion coefficient, thus determining the folding timescales of even millisecond folding proteins and hints at possible structural origins for the shallow landscape.


Assuntos
Dobramento de Proteína , Proteínas , Difusão , Fricção , Cinética , Termodinâmica
4.
J Inorg Biochem ; 210: 111169, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32679460

RESUMO

Classical molecular dynamic simulations and density functional theory are used to unveil the interaction of aluminum with various phosphorylated derivatives of the fragment KSPVPKSPVEEKG (NF13), a major multiphosphorylation domain of human neurofilament medium (NFM). Our calculations reveal the rich coordination chemistry of the resultant structures with a clear tendency of aluminum to form multidentate structures, acting as a bridging agent between different sidechains and altering the local secondary structure around the binding site. Our evaluation of binding energies allows us to determine that phosphorylation has an increase in the affinity of these peptides towards aluminum, although the interaction is not as strong as well-known chelators of aluminum in biological systems. Finally, the presence of hydroxides in the first solvation layer has a clear damping effect on the binding affinities. Our results help in elucidating the potential structures than can be formed between this exogenous neurotoxic metal and key sequences for the formation of neurofilament tangles, which are behind of some of the most important degenerative diseases.

5.
Phys Chem Chem Phys ; 22(15): 8118-8127, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32242581

RESUMO

Many intrinsically disordered proteins (IDPs) are involved in complex signalling networks inside the cell. Their particular binding modes elicit different types of responses that can be subtly regulated. Here we study the binding of two disordered transactivation domains from proteins HIF-1α and CITED2, whose binding to the TAZ1 domain of CBP is critical for the hypoxic response. Experiments have shown that both IDPs compete for their shared partner, and that this competition is mediated by the formation of a ternary intermediate state. Here we use computer simulations with a coarse-grained model to provide a detailed molecular description of this intermediate. We find that the conserved LP(Q/E)L motif may have a critical role in the displacement of HIF-1α by CITED2 and show a possible mechanism for the transition from the intermediate to the bound state. We also explore the role of TAZ1 dynamics in the binding. The results of our simulations are consistent with many of the experimental observations and provide a detailed view of the emergent properties in the complex binding of these IDPs.


Assuntos
Simulação por Computador , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Modelos Moleculares , Domínios Proteicos , Proteínas Repressoras/química , Transativadores/química , Motivos de Aminoácidos , Ligação Proteica , Estrutura Quaternária de Proteína
6.
Nat Commun ; 10(1): 5828, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862885

RESUMO

Cells remodel their structure in response to mechanical strain. However, how mechanical forces are translated into biochemical signals that coordinate the structural changes observed at the plasma membrane (PM) and the underlying cytoskeleton during mechanoadaptation is unclear. Here, we show that PM mechanoadaptation is controlled by a tension-sensing pathway composed of c-Abl tyrosine kinase and membrane curvature regulator FBP17. FBP17 is recruited to caveolae to induce the formation of caveolar rosettes. FBP17 deficient cells have reduced rosette density, lack PM tension buffering capacity under osmotic shock, and cannot adapt to mechanical strain. Mechanistically, tension is transduced to the FBP17 F-BAR domain by direct phosphorylation mediated by c-Abl, a mechanosensitive molecule. This modification inhibits FBP17 membrane bending activity and releases FBP17-controlled inhibition of mDia1-dependent stress fibers, favoring membrane adaptation to increased tension. This mechanoprotective mechanism adapts the cell to changes in mechanical tension by coupling PM and actin cytoskeleton remodeling.


Assuntos
Cavéolas/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Mecanotransdução Celular , Proteínas Proto-Oncogênicas c-abl/metabolismo , Fibras de Estresse/metabolismo , Cavéolas/ultraestrutura , Proteínas de Ligação a Ácido Graxo/genética , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Microscopia Eletrônica , Fosforilação , RNA Interferente Pequeno/metabolismo , Fibras de Estresse/ultraestrutura , Estresse Mecânico
7.
J Chem Inf Model ; 59(9): 3625-3629, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31423789

RESUMO

Markov state models (MSMs) have become one of the most important techniques for understanding biomolecular transitions from classical molecular dynamics (MD) simulations. MSMs provide a systematized way of accessing the long time kinetics of the system of interest from the short-time scale microscopic transitions observed in simulation trajectories. At the same time, they provide a consistent description of the equilibrium and dynamical properties of the system of interest, and they are ideally suited for comparisons against experiment. A few software packages exist for building MSMs, which have been widely adopted. Here we introduce MasterMSM, a new Python package that uses the master equation formulation of MSMs and provides a number of new algorithms for building and analyzing these models. We demonstrate some of the most distinctive features of the package, including the estimation of rates, definition of core-sets for transition based assignment of states, the estimation of committors and fluxes, and the sensitivity analysis of the emerging networks. The package is available at https://github.com/daviddesancho/MasterMSM .


Assuntos
Cadeias de Markov , Simulação de Dinâmica Molecular , Software , Algoritmos , Cinética
8.
J Phys Chem B ; 122(49): 11147-11154, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30129367

RESUMO

The analysis and interpretation of single molecule force spectroscopy (smFS) experiments is often complicated by hidden effects from the measuring device. Here we investigate these effects in our recent smFS experiments on the ultrafast folding protein gpW, which has been previously shown to fold without crossing a free energy barrier in the absence of force (i.e., downhill folding). Using atomic force microscopy (AFM) smFS experiments, we found that a very small force of ∼5 pN brings gpW near its unfolding midpoint and results in two-state (un)folding patterns that indicate the emergence of a force-induced free energy barrier. The change in the folding regime is concomitant with a 30,000-fold slowdown of the folding and unfolding times, from a few microseconds that it takes gpW to (un)fold at the midpoint temperature to seconds in the AFM. These results are puzzling because the barrier induced by force in the folding free energy landscape of gpW is far too small to account for such a difference in time scales. Here we use recently developed theoretical methods to resolve the origin of the strikingly slow dynamics of gpW under mechanical force. We find that, while the AFM experiments correctly capture the equilibrium distance distribution, the measured dynamics are entirely controlled by the response of the cantilever and polyprotein linker, which is much slower than the protein conformational dynamics. This interpretation is likely applicable to the folding of other small biomolecules in smFS experiments, and becomes particularly important in the case of systems with fast folding dynamics and small free energy barriers, and for instruments with slow response times.


Assuntos
Dobramento de Proteína , Proteínas/química , Fenômenos Mecânicos , Microscopia de Força Atômica , Conformação Proteica , Temperatura
9.
Nat Commun ; 9(1): 2758, 2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-30013059

RESUMO

Uropathogenic Escherichia coli attach to tissues using pili type 1. Each pilus is composed by thousands of coiled FimA domains followed by the domains of the tip fibrillum, FimF-FimG-FimH. The domains are linked by non-covalent ß-strands that must resist mechanical forces during attachment. Here, we use single-molecule force spectroscopy to measure the mechanical contribution of each domain to the stability of the pilus and monitor the oxidative folding mechanism of a single Fim domain assisted by periplasmic FimC and the oxidoreductase DsbA. We demonstrate that pilus domains bear high mechanical stability following a hierarchy by which domains close to the tip are weaker than those close to or at the pilus rod. During folding, this remarkable stability is achieved by the intervention of DsbA that not only forms strategic disulfide bonds but also serves as a chaperone assisting the folding of the domains.


Assuntos
Adesinas de Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/genética , Isomerases de Dissulfetos de Proteínas/química , Escherichia coli Uropatogênica/genética , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Sítios de Ligação , Clonagem Molecular , Dissulfetos/química , Dissulfetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/ultraestrutura , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Microscopia de Força Atômica , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/ultraestrutura
10.
J Chem Phys ; 148(21): 214107, 2018 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-29884049

RESUMO

Molecular dynamics (MD) simulations can model the interactions between macromolecules with high spatiotemporal resolution but at a high computational cost. By combining high-throughput MD with Markov state models (MSMs), it is now possible to obtain long time-scale behavior of small to intermediate biomolecules and complexes. To model the interactions of many molecules at large length scales, particle-based reaction-diffusion (RD) simulations are more suitable but lack molecular detail. Thus, coupling MSMs and RD simulations (MSM/RD) would be highly desirable, as they could efficiently produce simulations at large time and length scales, while still conserving the characteristic features of the interactions observed at atomic detail. While such a coupling seems straightforward, fundamental questions are still open: Which definition of MSM states is suitable? Which protocol to merge and split RD particles in an association/dissociation reaction will conserve the correct bimolecular kinetics and thermodynamics? In this paper, we make the first step toward MSM/RD by laying out a general theory of coupling and proposing a first implementation for association/dissociation of a protein with a small ligand (A + B ⇌ C). Applications on a toy model and CO diffusion into the heme cavity of myoglobin are reported.

12.
Chem Soc Rev ; 47(10): 3558-3573, 2018 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-29473060

RESUMO

Although much of our understanding of protein folding comes from studies of isolated protein domains in bulk, in the cellular environment the intervention of external molecular machines is essential during the protein life cycle. During the past decade single molecule force spectroscopy techniques have been extremely useful to deepen our understanding of these interventional molecular processes, as they allow for monitoring and manipulating mechanochemical events in individual protein molecules. Here, we review some of the critical steps in the protein life cycle, starting with the biosynthesis of the nascent polypeptide chain in the ribosome, continuing with the folding supported by chaperones and the translocation into different cell compartments, and ending with proteolysis in the proteasome. Along these steps, proteins experience molecular forces often combined with chemical transformations, affecting their folding and structure, which are measured or mimicked in the laboratory by the application of force with a single molecule apparatus. These mechanochemical reactions can potentially be used as targets for fighting against diseases. Inspired by these insightful experiments, we devise an outlook on the emerging field of mechanopharmacology, which reflects an alternative paradigm for drug design.


Assuntos
Proteínas/química , Estresse Mecânico , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteínas/metabolismo
13.
Phys Chem Chem Phys ; 19(42): 28512-28516, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29044256

RESUMO

Proteins that fold upon binding to their partners exhibit complex binding behavior such as induced-fit. But the connections between the folding mechanism and the binding mode remain unknown. Here we focus on the high affinity complex between the physiologically and marginally unstable, fast folder PSBD and the E1 subunit of pyruvate dehydrogenase. Using coarse-grained simulations we investigate the binding to E1 of a partially disordered PSBD under two folding scenarios: two-state and downhill. Our simulations show that induced-fit binding requires that PSBD folds-unfolds in the downhill folding regime. In contrast, a two-state folding PSBD must fold completely before it binds. The reason is that effective coupling between folding and binding involves partially folded conformations, which are only sufficiently populated under the downhill folding regime. Our results establish a direct mechanistic link between complex binding and downhill folding, supporting the idea that PSBD operates functionally as a conformational rheostat.


Assuntos
Ligação Proteica , Dobramento de Proteína , Fenômenos Biofísicos , Cinética , Proteínas Serina-Treonina Quinases/química , Termodinâmica
14.
Nat Struct Mol Biol ; 24(8): 652-657, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28671667

RESUMO

The sarcomere-based structure of muscles is conserved among vertebrates; however, vertebrate muscle physiology is extremely diverse. A molecular explanation for this diversity and its evolution has not been proposed. We use phylogenetic analyses and single-molecule force spectroscopy (smFS) to investigate the mechanochemical evolution of titin, a giant protein responsible for the elasticity of muscle filaments. We resurrect eight-domain fragments of titin corresponding to the common ancestors to mammals, sauropsids, and tetrapods, which lived 105-356 Myr ago, and compare them with titin fragments from some of their modern descendants. We demonstrate that the resurrected titin molecules are rich in disulfide bonds and display high mechanical stability. These mechanochemical elements have changed over time, creating a paleomechanical trend that seems to correlate with animal body size, allowing us to estimate the sizes of extinct species. We hypothesize that mechanical adjustments in titin contributed to physiological changes that allowed the muscular development and diversity of modern tetrapods.


Assuntos
Fenômenos Químicos , Conectina/genética , Conectina/metabolismo , Evolução Molecular , Fenômenos Mecânicos , Animais , Dissulfetos/análise , Filogenia , Análise Espectral , Vertebrados
15.
J Biol Chem ; 292(32): 13374-13380, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28642368

RESUMO

Disulfide bonds play a crucial role in proteins, modulating their stability and constraining their conformational dynamics. A particularly important case is that of proteins that need to withstand forces arising from their normal biological function and that are often disulfide bonded. However, the influence of disulfides on the overall mechanical stability of proteins is poorly understood. Here, we used single-molecule force spectroscopy (smFS) to study the role of disulfide bonds in different mechanical proteins in terms of their unfolding forces. For this purpose, we chose the pilus protein FimG from Gram-negative bacteria and a disulfide-bonded variant of the I91 human cardiac titin polyprotein. Our results show that disulfide bonds can alter the mechanical stability of proteins in different ways depending on the properties of the system. Specifically, disulfide-bonded FimG undergoes a 30% increase in its mechanical stability compared with its reduced counterpart, whereas the unfolding force of I91 domains experiences a decrease of 15% relative to the WT form. Using a coarse-grained simulation model, we rationalized that the increase in mechanical stability of FimG is due to a shift in the mechanical unfolding pathway. The simple topology-based explanation suggests a neutral effect in the case of titin. In summary, our results indicate that disulfide bonds in proteins act in a context-dependent manner rather than simply as mechanical lockers, underscoring the importance of considering disulfide bonds both computationally and experimentally when studying the mechanical properties of proteins.


Assuntos
Conectina/química , Cisteína/química , Cistina/química , Proteínas de Escherichia coli/química , Proteínas de Fímbrias/química , Modelos Moleculares , Substituição de Aminoácidos , Conectina/genética , Conectina/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Desdobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula
16.
Nat Chem ; 9(1): 88-95, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27995927

RESUMO

FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.


Assuntos
Hidrogênio/química , Hidrogenase/química , Oxigênio/química , Catálise , Clostridium/enzimologia , Difusão , Técnicas Eletroquímicas , Hidrogenase/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Teoria Quântica
17.
J Phys Chem Lett ; 7(19): 3798-3803, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27626458

RESUMO

Most experimentally well-characterized single domain proteins of less than 100 residues have been found to be two-state folders. That is, only two distinct populations can explain both equilibrium and kinetic measurements. Results from single molecule force spectroscopy, where a protein is unfolded by applying a mechanical pulling force to its ends, have largely confirmed this description for proteins found to be two-state in ensemble experiments. Recently, however, stable intermediates have been reported in mechanical unfolding experiments on a cold-shock protein previously found to be a prototypical two-state folder. Here, we tackle this discrepancy using free energy landscapes and Markov state models derived from coarse-grained molecular simulations. We show that protein folding intermediates can be selectively stabilized by the pulling force and that the populations of these intermediates vary in a force-dependent manner. Our model qualitatively captures the experimental results and suggests a possible origin of the apparent discrepancy.


Assuntos
Proteínas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas e Peptídeos de Choque Frio/química , Proteínas e Peptídeos de Choque Frio/metabolismo , Cinética , Cadeias de Markov , Microscopia de Força Atômica , Dobramento de Proteína , Desdobramento de Proteína , Proteínas/metabolismo , Termodinâmica , Thermotoga maritima/metabolismo
18.
J Mol Biol ; 428(21): 4245-4257, 2016 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-27639437

RESUMO

One of the major challenges in modern biophysics is observing and understanding conformational changes during complex molecular processes, from the fundamental protein folding to the function of molecular machines. Single-molecule techniques have been one of the major driving forces of the huge progress attained in the last few years. Recent advances in resolution of the experimental setups, aided by theoretical developments and molecular dynamics simulations, have revealed a much higher degree of complexity inside these molecular processes than previously reported using traditional ensemble measurements. This review sums up the evolution of these developments and gives an outlook on prospective discoveries.


Assuntos
Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Imagem Individual de Molécula/métodos
19.
J Phys Chem Lett ; 7(6): 1028-34, 2016 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-26947615

RESUMO

Recent experiments have revealed an unexpected deviation from a first power dependence of protein relaxation times on solvent viscosity, an effect that has been attributed to "internal friction". One clear source of internal friction in protein dynamics is the isomerization of dihedral angles. A key outstanding question is whether the global folding barrier height influences the measured internal friction, based on the observation that the folding rates of fast-folding proteins, with smaller folding free energy barriers, tend to exhibit larger internal friction. Here, by studying two alanine-based peptides, we find that systematic variation of global folding barrier heights has little effect on the internal friction for folding rates. On the other hand, increasing local torsion angle barriers leads to increased internal friction, which is consistent with solvent memory effects being the origin of the viscosity dependence. Thus, it appears that local torsion transitions determine the viscosity dependence of the diffusion coefficient on the global coordinate and, in turn, internal friction effects on the folding rate.


Assuntos
Dobramento de Proteína , Proteínas/química , Dipeptídeos/química , Transferência de Energia , Fricção , Isomerismo , Modelos Químicos , Estrutura Molecular , Estrutura Secundária de Proteína , Viscosidade
20.
Sci Rep ; 6: 21899, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26915629

RESUMO

High-resolution microscopy techniques have been extensively used to investigate the structure of soft, biological matter at the nanoscale, from very thin membranes to small objects, like viruses. Electron microscopy techniques allow for obtaining extraordinary resolution by averaging signals from multiple identical structures. In contrast, atomic force microscopy (AFM) collects data from single entities. Here, it is possible to finely modulate the interaction with the samples, in order to be sensitive to their top surface, avoiding mechanical deformations. However, most biological surfaces are highly curved, such as fibers or tubes, and ultimate details of their surface are in the vicinity of steep height variations. This limits lateral resolution, even when sharp probes are used. We overcome this problem by using multifrequency force microscopy on a textbook example, the Tobacco Mosaic Virus (TMV). We achieved unprecedented resolution in local maps of amplitude and phase shift of the second excited mode, recorded together with sample topography. Our data, which combine multifrequency imaging and Fourier analysis, confirm the structure deduced from averaging techniques (XRD, cryoEM) for surface features of single virus particles, down to the helical pitch of the coat protein subunits, 2.3 nm. Remarkably, multifrequency AFM images do not require any image postprocessing.


Assuntos
Microscopia de Força Atômica/métodos , Vírus do Mosaico do Tabaco/ultraestrutura , Vírion/ultraestrutura
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