Characterization of mechanical unfolding intermediates of membrane proteins by coarse grained molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Yamada, Tatsuya; Mitaku, Shigeki; Yamato, Takahisa

2018-01-01

Single-molecule force spectroscopy by atomic force microscopy allows us to get insight into the mechanical unfolding of membrane proteins, and a typical experiment exhibits characteristic patterns on the force distance curves. The origin of these patterns, however, has not been fully understood yet. We performed coarse-grained simulation of the forced unfolding of halorodopsin, reproduced the characteristic features of the experimental force distance curves. A further examination near the membrane-water interface indicated the existence of a motif for the force peak formation, i.e., the occurrence of hydrophobic residues in the upper interface region and hydrophilic residues below the lower interface region.

Digital force-feedback for protein unfolding experiments using atomic force microscopy

NASA Astrophysics Data System (ADS)

Bippes, Christian A.; Janovjak, Harald; Kedrov, Alexej; Muller, Daniel J.

2007-01-01

Since its invention in the 1990s single-molecule force spectroscopy has been increasingly applied to study protein (un-)folding, cell adhesion, and ligand-receptor interactions. In most force spectroscopy studies, the cantilever of an atomic force microscope (AFM) is separated from a surface at a constant velocity, thus applying an increasing force to folded bio-molecules or bio-molecular bonds. Recently, Fernandez and co-workers introduced the so-called force-clamp technique. Single proteins were subjected to a defined constant force allowing their life times and life time distributions to be directly measured. Up to now, the force-clamping was performed by analogue PID controllers, which require complex additional hardware and might make it difficult to combine the force-feedback with other modes such as constant velocity. These points may be limiting the applicability and versatility of this technique. Here we present a simple, fast, and all-digital (software-based) PID controller that yields response times of a few milliseconds in combination with a commercial AFM. We demonstrate the performance of our feedback loop by force-clamp unfolding of single Ig27 domains of titin and the membrane proteins bacteriorhodopsin (BR) and the sodium/proton antiporter NhaA.

Comparing Molecular Dynamics Force Fields in the Essential Subspace

PubMed Central

Gomez-Puertas, Paulino; Boomsma, Wouter; Lindorff-Larsen, Kresten

2015-01-01

The continued development and utility of molecular dynamics simulations requires improvements in both the physical models used (force fields) and in our ability to sample the Boltzmann distribution of these models. Recent developments in both areas have made available multi-microsecond simulations of two proteins, ubiquitin and Protein G, using a number of different force fields. Although these force fields mostly share a common mathematical form, they differ in their parameters and in the philosophy by which these were derived, and previous analyses showed varying levels of agreement with experimental NMR data. To complement the comparison to experiments, we have performed a structural analysis of and comparison between these simulations, thereby providing insight into the relationship between force-field parameterization, the resulting ensemble of conformations and the agreement with experiments. In particular, our results show that, at a coarse level, many of the motional properties are preserved across several, though not all, force fields. At a finer level of detail, however, there are distinct differences in both the structure and dynamics of the two proteins, which can, together with comparison with experimental data, help to select force fields for simulations of proteins. A noteworthy observation is that force fields that have been reparameterized and improved to provide a more accurate energetic description of the balance between helical and coil structures are difficult to distinguish from their “unbalanced” counterparts in these simulations. This observation implies that simulations of stable, folded proteins, even those reaching 10 microseconds in length, may provide relatively little information that can be used to modify torsion parameters to achieve an accurate balance between different secondary structural elements. PMID:25811178

Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

NASA Astrophysics Data System (ADS)

Kumar, Nitin; Gardel, Margaret

Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

Force-dependent switch in protein unfolding pathways and transition-state movements

PubMed Central

Zhuravlev, Pavel I.; Hinczewski, Michael; Chakrabarti, Shaon; Marqusee, Susan; Thirumalai, D.

2016-01-01

Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, ku(f), as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a log⁡ku(f) plot. Similar observations were reported for other proteins for the unfolding rate ku([C]). These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [C] is increased from a low to a high value. We provide a unified theory demonstrating that if log⁡ku as a function of a perturbation (f or [C]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in log⁡ku(f) to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants. PMID:26818842

Force-dependent switch in protein unfolding pathways and transition-state movements.

PubMed

Zhuravlev, Pavel I; Hinczewski, Michael; Chakrabarti, Shaon; Marqusee, Susan; Thirumalai, D

2016-02-09

Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, [Formula: see text], as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a [Formula: see text] plot. Similar observations were reported for other proteins for the unfolding rate [Formula: see text]. These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [Formula: see text] is increased from a low to a high value. We provide a unified theory demonstrating that if [Formula: see text] as a function of a perturbation (f or [Formula: see text]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in [Formula: see text] to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants.

A force-based, parallel assay for the quantification of protein-DNA interactions.

PubMed

Limmer, Katja; Pippig, Diana A; Aschenbrenner, Daniela; Gaub, Hermann E

2014-01-01

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

The force exerted by the membrane potential during protein import into the mitochondrial matrix

NASA Technical Reports Server (NTRS)

Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

2004-01-01

The force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated on the basis of continuum electrostatics. The force is found to vary from 3.0 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 6.5 to 12 A, its measured range. In the present model, the decrease in force with increasing pore width arises from the shielding effect of water. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a purely membranous pore without water gives a force of 3.2 pN per unit charge, which should represent an upper limit. When applied to mitochondrial import experiments on the protein barnase, these results imply that forces between 11 +/- 2 pN and 13.5 +/- 2.5 pN catalyze the unfolding of barnase in those experiments. A comparison of these results with unfolding forces measured using atomic force microscopy is made.

Intrinsically Disordered Protein Specific Force Field CHARMM36IDPSFF.

PubMed

Liu, Hao; Song, Dong; Lu, Hui; Luo, Ray; Chen, Hai-Feng

2018-05-28

Intrinsically disordered proteins (IDPs) are closely related to various human diseases. Because IDPs lack certain tertiary structure, it is difficult to use X-ray and NMR methods to measure their structures. Therefore, molecular dynamics simulation is a useful tool to study the conformer distribution of IDPs. However, most generic protein force fields were found to be insufficient in simulations of IDPs. Here we report our development for the CHARMM community. Our residue-specific IDP force field (CHARMM36IDPSFF) was developed based on the base generic force field with CMAP corrections of for all 20 naturally occurring amino acids. Multiple tests show that the simulated chemical shifts with the newly developed force field are in quantitative agreement with NMR experiment and are more accurate than the base generic force field. Comparison of J-couplings with previous work shows that CHARMM36IDPSFF and its corresponding base generic force field have their own advantages. In addition, CHARMM36IDPSFF simulations also agree with experiment for SAXS profiles and radii of gyration of IDPs. Detailed analysis shows that CHARMM36IDPSFF can sample more diverse and disordered conformers. These findings confirm that the newly developed force field can improve the balance of accuracy and efficiency for the conformer sampling of IDPs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Toward optimized potential functions for protein-protein interactions in aqueous solutions: osmotic second virial coefficient calculations using the MARTINI coarse-grained force field

PubMed Central

Stark, Austin C.; Andrews, Casey T.

2013-01-01

Coarse-grained (CG) simulation methods are now widely used to model the structure and dynamics of large biomolecular systems. One important issue for using such methods – especially with regard to using them to model, for example, intracellular environments – is to demonstrate that they can reproduce experimental data on the thermodynamics of protein-protein interactions in aqueous solutions. To examine this issue, we describe here simulations performed using the popular coarse-grained MARTINI force field, aimed at computing the thermodynamics of lysozyme and chymotrypsinogen self-interactions in aqueous solution. Using molecular dynamics simulations to compute potentials of mean force between a pair of protein molecules, we show that the original parameterization of the MARTINI force field is likely to significantly overestimate the strength of protein-protein interactions to the extent that the computed osmotic second virial coefficients are orders of magnitude more negative than experimental estimates. We then show that a simple down-scaling of the van der Waals parameters that describe the interactions between protein pseudo-atoms can bring the simulated thermodynamics into much closer agreement with experiment. Overall, the work shows that it is feasible to test explicit-solvent CG force fields directly against thermodynamic data for proteins in aqueous solutions, and highlights the potential usefulness of osmotic second virial coefficient measurements for fully parameterizing such force fields. PMID:24223529

Toward optimized potential functions for protein-protein interactions in aqueous solutions: osmotic second virial coefficient calculations using the MARTINI coarse-grained force field.

PubMed

Stark, Austin C; Andrews, Casey T; Elcock, Adrian H

2013-09-10

Coarse-grained (CG) simulation methods are now widely used to model the structure and dynamics of large biomolecular systems. One important issue for using such methods - especially with regard to using them to model, for example, intracellular environments - is to demonstrate that they can reproduce experimental data on the thermodynamics of protein-protein interactions in aqueous solutions. To examine this issue, we describe here simulations performed using the popular coarse-grained MARTINI force field, aimed at computing the thermodynamics of lysozyme and chymotrypsinogen self-interactions in aqueous solution. Using molecular dynamics simulations to compute potentials of mean force between a pair of protein molecules, we show that the original parameterization of the MARTINI force field is likely to significantly overestimate the strength of protein-protein interactions to the extent that the computed osmotic second virial coefficients are orders of magnitude more negative than experimental estimates. We then show that a simple down-scaling of the van der Waals parameters that describe the interactions between protein pseudo-atoms can bring the simulated thermodynamics into much closer agreement with experiment. Overall, the work shows that it is feasible to test explicit-solvent CG force fields directly against thermodynamic data for proteins in aqueous solutions, and highlights the potential usefulness of osmotic second virial coefficient measurements for fully parameterizing such force fields.

Mechanical Network in Titin Immunoglobulin from Force Distribution Analysis

PubMed Central

Wilmanns, Matthias; Gräter, Frauke

2009-01-01

The role of mechanical force in cellular processes is increasingly revealed by single molecule experiments and simulations of force-induced transitions in proteins. How the applied force propagates within proteins determines their mechanical behavior yet remains largely unknown. We present a new method based on molecular dynamics simulations to disclose the distribution of strain in protein structures, here for the newly determined high-resolution crystal structure of I27, a titin immunoglobulin (IG) domain. We obtain a sparse, spatially connected, and highly anisotropic mechanical network. This allows us to detect load-bearing motifs composed of interstrand hydrogen bonds and hydrophobic core interactions, including parts distal to the site to which force was applied. The role of the force distribution pattern for mechanical stability is tested by in silico unfolding of I27 mutants. We then compare the observed force pattern to the sparse network of coevolved residues found in this family. We find a remarkable overlap, suggesting the force distribution to reflect constraints for the evolutionary design of mechanical resistance in the IG family. The force distribution analysis provides a molecular interpretation of coevolution and opens the road to the study of the mechanism of signal propagation in proteins in general. PMID:19282960

Following aptamer-ricin specific binding by single molecule recognition and force spectroscopy measurements

USDA-ARS?s Scientific Manuscript database

The atomic force microscope (AFM) recognition and dynamic force spectroscopy (DFS) experiments provide both morphology and interaction information of the aptamer and protein, which can be used for the future study on the thermodynamics and kinetics properties of ricin-aptamer/antibody interactions. ...

How well do force fields capture the strength of salt bridges in proteins?

PubMed Central

Ahmed, Mustapha Carab; Papaleo, Elena

2018-01-01

Salt bridges form between pairs of ionisable residues in close proximity and are important interactions in proteins. While salt bridges are known to be important both for protein stability, recognition and regulation, we still do not have fully accurate predictive models to assess the energetic contributions of salt bridges. Molecular dynamics simulation is one technique that may be used study the complex relationship between structure, solvation and energetics of salt bridges, but the accuracy of such simulations depends on the force field used. We have used NMR data on the B1 domain of protein G (GB1) to benchmark molecular dynamics simulations. Using enhanced sampling simulations, we calculated the free energy of forming a salt bridge for three possible lysine-carboxylate ionic interactions in GB1. The NMR experiments showed that these interactions are either not formed, or only very weakly formed, in solution. In contrast, we show that the stability of the salt bridges is overestimated, to different extents, in simulations of GB1 using seven out of eight commonly used combinations of fixed charge force fields and water models. We also find that the Amber ff15ipq force field gives rise to weaker salt bridges in good agreement with the NMR experiments. We conclude that many force fields appear to overstabilize these ionic interactions, and that further work may be needed to refine our ability to model quantitatively the stability of salt bridges through simulations. We also suggest that comparisons between NMR experiments and simulations will play a crucial role in furthering our understanding of this important interaction.

Direct Observation of Markovian Behavior of the Mechanical Unfolding of Individual Proteins

PubMed Central

Cao, Yi; Kuske, Rachel; Li, Hongbin

2008-01-01

Single-molecule force-clamp spectroscopy is a valuable tool to analyze unfolding kinetics of proteins. Previous force-clamp spectroscopy experiments have demonstrated that the mechanical unfolding of ubiquitin deviates from the generally assumed Markovian behavior and involves the features of glassy dynamics. Here we use single molecule force-clamp spectroscopy to study the unfolding kinetics of a computationally designed fast-folding mutant of the small protein GB1, which shares a similar β-grasp fold as ubiquitin. By treating the mechanical unfolding of polyproteins as the superposition of multiple identical Poisson processes, we developed a simple stochastic analysis approach to analyze the dwell time distribution of individual unfolding events in polyprotein unfolding trajectories. Our results unambiguously demonstrate that the mechanical unfolding of NuG2 fulfills all criteria of a memoryless Markovian process. This result, in contrast with the complex mechanical unfolding behaviors observed for ubiquitin, serves as a direct experimental demonstration of the Markovian behavior for the mechanical unfolding of a protein and reveals the complexity of the unfolding dynamics among structurally similar proteins. Furthermore, we extended our method into a robust and efficient pseudo-dwell-time analysis method, which allows one to make full use of all the unfolding events obtained in force-clamp experiments without categorizing the unfolding events. This method enabled us to measure the key parameters characterizing the mechanical unfolding energy landscape of NuG2 with improved precision. We anticipate that the methods demonstrated here will find broad applications in single-molecule force-clamp spectroscopy studies for a wide range of proteins. PMID:18375518

Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA–protein complexes

PubMed Central

McAndrew, Christopher P.; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L.; Pegg, Ian L.; Sarkar, Abhijit

2016-01-01

We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA–protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether’s extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule’s force response. Using a calibration technique based on Stokes’ drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level. PMID:26757808

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sotomayor, Marcos

Hair cell mechanotransduction happens in tens of microseconds, involves forces of a few picoNewtons, and is mediated by nanometer-scale molecular conformational changes. As proteins involved in this process become identified and their high resolution structures become available, multiple tools are being used to explore their “single-molecule responses” to force. Optical tweezers and atomic force microscopy offer exquisite force and extension resolution, but cannot reach the high loading rates expected for high frequency auditory stimuli. Molecular dynamics (MD) simulations can reach these fast time scales, and also provide a unique view of the molecular events underlying protein mechanics, but its predictionsmore » must be experimentally verified. Thus a combination of simulations and experiments might be appropriate to study the molecular mechanics of hearing. Here I review the basics of MD simulations and the different methods used to apply force and study protein mechanics in silico. Simulations of tip link proteins are used to illustrate the advantages and limitations of this method.« less

Kirkwood-Buff Approach Rescues Overcollapse of a Disordered Protein in Canonical Protein Force Fields.

PubMed

Mercadante, Davide; Milles, Sigrid; Fuertes, Gustavo; Svergun, Dmitri I; Lemke, Edward A; Gräter, Frauke

2015-06-25

Understanding the function of intrinsically disordered proteins is intimately related to our capacity to correctly sample their conformational dynamics. So far, a gap between experimentally and computationally derived ensembles exists, as simulations show overcompacted conformers. Increasing evidence suggests that the solvent plays a crucial role in shaping the ensembles of intrinsically disordered proteins and has led to several attempts to modify water parameters and thereby favor protein-water over protein-protein interactions. This study tackles the problem from a different perspective, which is the use of the Kirkwood-Buff theory of solutions to reproduce the correct conformational ensemble of intrinsically disordered proteins (IDPs). A protein force field recently developed on such a basis was found to be highly effective in reproducing ensembles for a fragment from the FG-rich nucleoporin 153, with dimensions matching experimental values obtained from small-angle X-ray scattering and single molecule FRET experiments. Kirkwood-Buff theory presents a complementary and fundamentally different approach to the recently developed four-site TIP4P-D water model, both of which can rescue the overcollapse observed in IDPs with canonical protein force fields. As such, our study provides a new route for tackling the deficiencies of current protein force fields in describing protein solvation.

The binding domain of the HMGB1 inhibitor carbenoxolone: Theory and experiment

NASA Astrophysics Data System (ADS)

Mollica, Luca; Curioni, Alessandro; Andreoni, Wanda; Bianchi, Marco E.; Musco, Giovanna

2008-05-01

We present a combined computational and experimental study of the interaction of the Box A of the HMGB1 protein and carbenoxolone, an inhibitor of its pro-inflammatory activity. The computational approach consists of classical molecular dynamics (MD) simulations based on the GROMOS force field with quantum-refined (QRFF) atomic charges for the ligand. Experimental data consist of fluorescence intensities, chemical shift displacements, saturation transfer differences and intermolecular Nuclear Overhauser Enhancement signals. Good agreement is found between observations and the conformation of the ligand-protein complex resulting from QRFF-MD. In contrast, simple docking procedures and MD based on the unrefined force field provide models inconsistent with experiment. The ligand-protein binding is dominated by non-directional interactions.

Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy.

PubMed

Kedrov, Alexej; Janovjak, Harald; Sapra, K Tanuj; Müller, Daniel J

2007-01-01

Molecular interactions are the basic language of biological processes. They establish the forces interacting between the building blocks of proteins and other macromolecules, thus determining their functional roles. Because molecular interactions trigger virtually every biological process, approaches to decipher their language are needed. Single-molecule force spectroscopy (SMFS) has been used to detect and characterize different types of molecular interactions that occur between and within native membrane proteins. The first experiments detected and localized molecular interactions that stabilized membrane proteins, including how these interactions were established during folding of alpha-helical secondary structure elements into the native protein and how they changed with oligomerization, temperature, and mutations. SMFS also enables investigators to detect and locate molecular interactions established during ligand and inhibitor binding. These exciting applications provide opportunities for studying the molecular forces of life. Further developments will elucidate the origins of molecular interactions encoded in their lifetimes, interaction ranges, interplay, and dynamics characteristic of biological systems.

Optimized molecular dynamics force fields applied to the helix-coil transition of polypeptides.

PubMed

Best, Robert B; Hummer, Gerhard

2009-07-02

Obtaining the correct balance of secondary structure propensities is a central priority in protein force-field development. Given that current force fields differ significantly in their alpha-helical propensities, a correction to match experimental results would be highly desirable. We have determined simple backbone energy corrections for two force fields to reproduce the fraction of helix measured in short peptides at 300 K. As validation, we show that the optimized force fields produce results in excellent agreement with nuclear magnetic resonance experiments for folded proteins and short peptides not used in the optimization. However, despite the agreement at ambient conditions, the dependence of the helix content on temperature is too weak, a problem shared with other force fields. A fit of the Lifson-Roig helix-coil theory shows that both the enthalpy and entropy of helix formation are too small: the helix extension parameter w agrees well with experiment, but its entropic and enthalpic components are both only about half the respective experimental estimates. Our structural and thermodynamic analyses point toward the physical origins of these shortcomings in current force fields, and suggest ways to address them in future force-field development.

Evidence of protein-free homology recognition in magnetic bead force-extension experiments

NASA Astrophysics Data System (ADS)

O'Lee, D. J.; Danilowicz, C.; Rochester, C.; Kornyshev, A. A.; Prentiss, M.

2016-07-01

Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data.

Scrutinizing Molecular Mechanics Force Fields on the Submicrosecond Timescale with NMR Data

PubMed Central

Lange, Oliver F.; van der Spoel, David; de Groot, Bert L.

2010-01-01

Abstract Protein dynamics on the atomic level and on the microsecond timescale has recently become accessible from both computation and experiment. To validate molecular dynamics (MD) at the submicrosecond timescale against experiment we present microsecond MD simulations in 10 different force-field configurations for two globular proteins, ubiquitin and the gb3 domain of protein G, for which extensive NMR data is available. We find that the reproduction of the measured NMR data strongly depends on the chosen force field and electrostatics treatment. Generally, particle-mesh Ewald outperforms cut-off and reaction-field approaches. A comparison to measured J-couplings across hydrogen bonds suggests that there is room for improvement in the force-field description of hydrogen bonds in most modern force fields. Our results show that with current force fields, simulations beyond hundreds of nanoseconds run an increased risk of undergoing transitions to nonnative conformational states or will persist within states of high free energy for too long, thus skewing the obtained population frequencies. Only for the AMBER99sb force field have such transitions not been observed. Thus, our results have significance for the interpretation of data obtained with long MD simulations, for the selection of force fields for MD studies and for force-field development. We hope that this comprehensive benchmark based on NMR data applied to many popular MD force fields will serve as a useful resource to the MD community. Finally, we find that for gb3, the force-field AMBER99sb reaches comparable accuracy in back-calculated residual dipolar couplings and J-couplings across hydrogen bonds to ensembles obtained by refinement against NMR data. PMID:20643085

Development and Implementation of a Protein-Protein Binding Experiment to Teach Intermolecular Interactions in High School or Undergraduate Classrooms

ERIC Educational Resources Information Center

Johnson, Sadie M.; Javner, Cassidy; Hackel, Benjamin J.

2017-01-01

The goal of this study was to create an accessible, inexpensive, and engaging experiment to teach high school and undergraduate chemistry or biology students about intermolecular forces and how they contribute to the behavior of biomolecules. We developed an enzyme-linked immunosorbent assay (ELISA) to probe specific structure-function…

The elastic free energy of a tandem modular protein under force.

PubMed

Valle-Orero, Jessica; Eckels, Edward C; Stirnemann, Guillaume; Popa, Ionel; Berkovich, Ronen; Fernandez, Julio M

2015-05-01

Recent studies have provided a theoretical framework for including entropic elasticity in the free energy landscape of proteins under mechanical force. Accounting for entropic elasticity using polymer physics models has helped explain the hopping behavior seen in single molecule experiments in the low force regime. Here, we expand on the construction of the free energy of a single protein domain under force proposed by Berkovich et al. to provide a free energy landscape for N tandem domains along a continuous polypeptide. Calculation of the free energy of individual domains followed by their concatenation provides a continuous free energy landscape whose curvature is dominated by the worm-like chain at forces below 20 pN. We have validated our free energy model using Brownian dynamics and reproduce key features of protein folding. This free energy model can predict the effects of changes in the elastic properties of a multidomain protein as a consequence of biological modifications such as phosphorylation or the formation of disulfide bonds. This work lays the foundations for the modeling of tissue elasticity, which is largely determined by the properties of tandem polyproteins. Copyright © 2015. Published by Elsevier Inc.

Effect of fuel concentration and force on collective transport by a team of dynein motors

PubMed Central

Takshak, Anjneya; Roy, Tanushree; Tandaiya, Parag

2016-01-01

Abstract Motor proteins are essential components of intracellular transport inside eukaryotic cells. These protein molecules use chemical energy obtained from hydrolysis of ATP to produce mechanical forces required for transporting cargos inside cells, from one location to another, in a directed manner. Of these motors, cytoplasmic dynein is structurally more complex than other motor proteins involved in intracellular transport, as it shows force and fuel (ATP) concentration dependent step‐size. Cytoplasmic dynein motors are known to work in a team during cargo transport and force generation. Here, we use a complete Monte‐Carlo model of single dynein constrained by in vitro experiments, which includes the effect of both force and ATP on stepping as well as detachment of motors under force. We then use our complete Monte‐Carlo model of single dynein motor to understand collective cargo transport by a team of dynein motors, such as dependence of cargo travel distance and velocity on applied force and fuel concentration. In our model, cargos pulled by a team of dynein motors do not detach rapidly under higher forces, confirming the experimental observation of longer persistence time of dynein team on microtubule under higher forces. PMID:27727483

Critical Comparison of Biomembrane Force Fields: Protein-Lipid Interactions at the Membrane Interface.

PubMed

Sandoval-Perez, Angelica; Pluhackova, Kristyna; Böckmann, Rainer A

2017-05-09

Molecular dynamics (MD) simulations offer the possibility to study biological processes at high spatial and temporal resolution often not reachable by experiments. Corresponding biomolecular force field parameters have been developed for a wide variety of molecules ranging from inorganic ligands and small organic molecules over proteins and lipids to nucleic acids. Force fields have typically been parametrized and validated on thermodynamic observables and structural characteristics of individual compounds, e.g. of soluble proteins or lipid bilayers. Less strictly, due to the added complexity and missing experimental data to compare to, force fields have hardly been tested on the properties of mixed systems, e.g. on protein-lipid systems. Their selection and combination for mixed systems is further complicated by the partially differing parametrization strategies. Additionally, the presence of other compounds in the system may shift the subtle balance of force field parameters. Here, we assessed the protein-lipid interactions as described in the four atomistic force fields GROMOS54a7, CHARMM36 and the two force field combinations Amber14sb/Slipids and Amber14sb/Lipid14. Four observables were compared, focusing on the membrane-water interface: the conservation of the secondary structure of transmembrane proteins, the positioning of transmembrane peptides relative to the lipid bilayer, the insertion depth of side chains of unfolded peptides absorbed at the membrane interface, and the ability to reproduce experimental insertion energies of Wimley-White peptides at the membrane interface. Significant differences between the force fields were observed that affect e.g. membrane insertion depths and tilting of transmembrane peptides.

Measurements of the driving forces of bio-motors using the fluctuation theorem

PubMed Central

Hayashi, Kumiko; Tanigawara, Mizue; Kishikawa, Jun-ichi

2012-01-01

The fluctuation theorem (FT), which is a recent achievement in non-equilibrium statistical mechanics, has been suggested to be useful for measuring the driving forces of motor proteins. As an example of this application, we performed single-molecule experiments on F1-ATPase, which is a rotary motor protein, in which we measured its rotary torque by taking advantage of FT. Because fluctuation is inherent nature in biological small systems and because FT is a non-destructive force measurement method using fluctuation, it will be applied to a wide range of biological small systems in future. PMID:27857609

Mechanism underlying bioinertness of self-assembled monolayers of oligo(ethyleneglycol)-terminated alkanethiols on gold: protein adsorption, platelet adhesion, and surface forces.

PubMed

Hayashi, Tomohiro; Tanaka, Yusaku; Koide, Yuki; Tanaka, Masaru; Hara, Masahiko

2012-08-07

The mechanism underlying the bioinertness of the self-assembled monolayers of oligo(ethylene glycol)-terminated alkanethiol (OEG-SAM) was investigated with protein adsorption experiments, platelet adhesion tests, and surface force measurements with an atomic force microscope (AFM). In this work, we performed systematic analysis with SAMs having various terminal groups (-OEG, -OH, -COOH, -NH(2), and -CH(3)). The results of the protein adsorption experiment by the quartz crystal microbalance (QCM) method suggested that having one EG unit and the neutrality of total charges of the terminal groups are essential for protein-resistance. In particular, QCM with energy dissipation analyses indicated that proteins absorb onto the OEG-SAM via a very weak interaction compared with other SAMs. Contrary to the protein resistance, at least three EG units as well as the charge neutrality of the SAM are found to be required for anti-platelet adhesion. When the identical SAMs were formed on both AFM probe and substrate, our force measurements revealed that only the OEG-SAMs possessing more than two EG units showed strong repulsion in the range of 4 to 6 nm. In addition, we found that the SAMs with other terminal groups did not exhibit such repulsion. The repulsion between OEG-SAMs was always observed independent of solution conditions [NaCl concentration (between 0 and 1 M) and pH (between 3 and 11)] and was not observed in solution mixed with ethanol, which disrupts the three-dimensional network of the water molecules. We therefore concluded that the repulsion originated from structured interfacial water molecules. Considering the correlation between the above results, we propose that the layer of the structured interfacial water with a thickness of 2 to 3 nm (half of the range of the repulsion observed in the surface force measurements) plays an important role in deterring proteins and platelets from adsorption or adhesion.

Enhanced sampling molecular dynamics simulation captures experimentally suggested intermediate and unfolded states in the folding pathway of Trp-cage miniprotein.

PubMed

Shao, Qiang; Shi, Jiye; Zhu, Weiliang

2012-09-28

The ability of molecular dynamics simulation to capturing the transient states within the folding pathway of protein is important to the understanding of protein folding mechanism. In the present study, the integrated-tempering-sampling molecular dynamics (ITS-MD) simulation was performed to investigate the transient states including intermediate and unfolded ones in the folding pathway of a miniprotein, Trp-cage. Three force fields (FF03, FF99SB, and FF96) were tested, and both intermediate and unfolded states with their characteristics in good agreement with experiments were observed during the simulations, which supports the hypothesis that observable intermediates might present in the folding pathway of small polypeptides. In addition, it was demonstrated that FF03 force field as combined with ITS-MD is in overall a more proper force field than the others in reproducing experimentally recorded properties in UVRS, ECD, and NMR, Photo-CIDNP NMR, and IR T-jump experiments, and the folding∕unfolding thermodynamics parameters, such as ΔG(U), ΔC(p), and ΔH(U) (T(m)). In summary, the present study showed that using suitable force field and energy sampling method, molecular dynamics simulation could capture the transient states within the folding pathway of protein which are consistent with the experimental measurements, and thus provide information of protein folding mechanism and thermodynamics.

Folding Free Energy Landscape of the Decapeptide Chignolin

NASA Astrophysics Data System (ADS)

Dou, Xianghua; Wang, Jihua

Chignolin is an artificially designed ten-residue (GYDPETGTWG) folded peptide, which is the smallest protein and provides a good template for protein folding. In this work, we completed four explicit water molecular dynamics simulations of Chignolin folding using GROMOS and OPLS-AA force fields from extended initial states without any experiment informations. The four-folding free energy landscapes of the peptide has been drawn. The folded state of Chignolin has been successfully predicated based on the free energy landscapes. The four independent simulations gave similar results. (i) The four free energy landscapes have common characters. They are fairly smooth, barrierless, funnel-like and downhill without intermediate state, which consists with the experiment. (ii) The different extended initial structures converge at similar folded structures with the lowest free energy under GROMOS and OPLS-AA force fields. In the GROMOS force field, the backbone RMSD of the folded structures from the NMR native structure of Chignolin is only 0.114 nm, which is a stable structure in this force field. In the OPLS-AA force field, the similar results have been obtained. In addition, the smallest RMSD structure is in better agreement with the NMR native structure but unlikely stable in the force field.

Exploring the Molecular Origins of Bio(in)compatibility: Adhesion Between Proteins and Individual Chains of Poly(ethylene oxide)

NASA Astrophysics Data System (ADS)

Rixman, Monica A.; Ortiz, Christine

2002-03-01

A critical determinant of the biocompatibility of implanted blood-contacting devices is the initial noncovalent adsorption of blood plasma proteins onto the biomaterial surface. Using high-resolution force spectroscopy, we have measured the complex intermolecular interaction forces between individual end-grafted PEO chains and a probe tip covalently bound with human serum albumin, the most abundant blood plasma protein in the human body. On approach, a long-range, nonlinear repulsive force is observed. Upon retraction, however, adhesion between the HSA probe tip and PEO chain occurs, which in many cases is strong enough to allow long-range adhesion and stretching of the individual PEO chains. The known PEO strain-induced conformational transition from the helical (ttg) to the planar (ttt) conformation is clearly observed and seen to shift to lower force values. Statistical analysis of adhesion data, comparison to a variety of control experiments, and theoretical modeling enable us to interpret these experimental results in terms of electrostatic interactions, hydrogen bonding, and steric forces.

The force-dependent mechanism of DnaK-mediated mechanical folding

PubMed Central

Perales-Calvo, Judit; Giganti, David; Stirnemann, Guillaume; Garcia-Manyes, Sergi

2018-01-01

It is well established that chaperones modulate the protein folding free-energy landscape. However, the molecular determinants underlying chaperone-mediated mechanical folding remain largely elusive, primarily because the force-extended unfolded conformation fundamentally differs from that characterized in biochemistry experiments. We use single-molecule force-clamp spectroscopy, combined with molecular dynamics simulations, to study the effect that the Hsp70 system has on the mechanical folding of three mechanically stiff model proteins. Our results demonstrate that, when working independently, DnaJ (Hsp40) and DnaK (Hsp70) work as holdases, blocking refolding by binding to distinct substrate conformations. Whereas DnaK binds to molten globule–like forms, DnaJ recognizes a cryptic sequence in the extended state in an unanticipated force-dependent manner. By contrast, the synergetic coupling of the Hsp70 system exhibits a marked foldase behavior. Our results offer unprecedented molecular and kinetic insights into the mechanisms by which mechanical force finely regulates chaperone binding, directly affecting protein elasticity. PMID:29487911

GRIFFIN: A versatile methodology for optimization of protein-lipid interfaces for membrane protein simulations

PubMed Central

Staritzbichler, René; Anselmi, Claudio; Forrest, Lucy R.; Faraldo-Gómez, José D.

2014-01-01

As new atomic structures of membrane proteins are resolved, they reveal increasingly complex transmembrane topologies, and highly irregular surfaces with crevices and pores. In many cases, specific interactions formed with the lipid membrane are functionally crucial, as is the overall lipid composition. Compounded with increasing protein size, these characteristics pose a challenge for the construction of simulation models of membrane proteins in lipid environments; clearly, that these models are sufficiently realistic bears upon the reliability of simulation-based studies of these systems. Here, we introduce GRIFFIN, which uses a versatile framework to automate and improve a widely-used membrane-embedding protocol. Initially, GRIFFIN carves out lipid and water molecules from a volume equivalent to that of the protein, so as to conserve the system density. In the subsequent optimization phase GRIFFIN adds an implicit grid-based protein force-field to a molecular dynamics simulation of the pre-carved membrane. In this force-field, atoms inside the implicit protein volume experience an outward force that will expel them from that volume, whereas those outside are subject to electrostatic and van-der-Waals interactions with the implicit protein. At each step of the simulation, these forces are updated by GRIFFIN and combined with the intermolecular forces of the explicit lipid-water system. This procedure enables the construction of realistic and reproducible starting configurations of the protein-membrane interface within a reasonable timeframe and with minimal intervention. GRIFFIN is a standalone tool designed to work alongside any existing molecular dynamics package, such as NAMD or GROMACS. PMID:24707227

Ab initio folding of proteins using all-atom discrete molecular dynamics

PubMed Central

Ding, Feng; Tsao, Douglas; Nie, Huifen; Dokholyan, Nikolay V.

2008-01-01

Summary Discrete molecular dynamics (DMD) is a rapid sampling method used in protein folding and aggregation studies. Until now, DMD was used to perform simulations of simplified protein models in conjunction with structure-based force fields. Here, we develop an all-atom protein model and a transferable force field featuring packing, solvation, and environment-dependent hydrogen bond interactions. Using the replica exchange method, we perform folding simulations of six small proteins (20–60 residues) with distinct native structures. In all cases, native or near-native states are reached in simulations. For three small proteins, multiple folding transitions are observed and the computationally-characterized thermodynamics are in quantitative agreement with experiments. The predictive power of all-atom DMD highlights the importance of environment-dependent hydrogen bond interactions in modeling protein folding. The developed approach can be used for accurate and rapid sampling of conformational spaces of proteins and protein-protein complexes, and applied to protein engineering and design of protein-protein interactions. PMID:18611374

New force replica exchange method and protein folding pathways probed by force-clamp technique.

PubMed

Kouza, Maksim; Hu, Chin-Kun; Li, Mai Suan

2008-01-28

We have developed a new extended replica exchange method to study thermodynamics of a system in the presence of external force. Our idea is based on the exchange between different force replicas to accelerate the equilibrium process. This new approach was applied to obtain the force-temperature phase diagram and other thermodynamical quantities of the three-domain ubiquitin. Using the C(alpha)-Go model and the Langevin dynamics, we have shown that the refolding pathways of single ubiquitin depend on which terminus is fixed. If the N end is fixed then the folding pathways are different compared to the case when both termini are free, but fixing the C terminal does not change them. Surprisingly, we have found that the anchoring terminal does not affect the pathways of individual secondary structures of three-domain ubiquitin, indicating the important role of the multidomain construction. Therefore, force-clamp experiments, in which one end of a protein is kept fixed, can probe the refolding pathways of a single free-end ubiquitin if one uses either the polyubiquitin or a single domain with the C terminus anchored. However, it is shown that anchoring one end does not affect refolding pathways of the titin domain I27, and the force-clamp spectroscopy is always capable to predict folding sequencing of this protein. We have obtained the reasonable estimate for unfolding barrier of ubiquitin, using the microscopic theory for the dependence of unfolding time on the external force. The linkage between residue Lys48 and the C terminal of ubiquitin is found to have the dramatic effect on the location of the transition state along the end-to-end distance reaction coordinate, but the multidomain construction leaves the transition state almost unchanged. We have found that the maximum force in the force-extension profile from constant velocity force pulling simulations depends on temperature nonlinearly. However, for some narrow temperature interval this dependence becomes linear, as have been observed in recent experiments.

Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

NASA Astrophysics Data System (ADS)

Wijeratne, Sithara S.; Botello, Eric; Yeh, Hui-Chun; Zhou, Zhou; Bergeron, Angela L.; Frey, Eric W.; Patel, Jay M.; Nolasco, Leticia; Turner, Nancy A.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-Hwa

2013-03-01

The mechanical force-induced activation of the adhesive protein von Willebrand factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (PVWF) multimers to bind platelets. Here, we showed that a pathological level of high shear stress exposure of PVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

ff14IDPs Force Field Improving the Conformation Sampling of Intrinsically Disordered Proteins

PubMed Central

Song, Dong; Wang, Wei; Ye, Wei; Ji, Dingjue; Luo, Ray; Chen, Hai-Feng

2017-01-01

Intrinsically disordered proteins (IDPs) are proteins which lack of specific tertiary structure and unable to fold spontaneously without the partner binding. These IDPs are found to associate with various diseases, such as diabetes, cancer, and neurodegenerative diseases. However, current widely used force fields, such as ff99SB, ff14SB, OPLS/AA, and Charmm27 are insufficient in sampling the conformational characters of IDPs. In this study, the CMAP method was used to correct the φ/ψ distributions of disorder-promoting amino acids. The simulation results show that the force filed parameters (ff14IDPs) can improve the φ/ψ distributions of the disorder-promoting amino acids, with RMSD less than 0.10% relative to the benchmark data of IDPs. Further test suggests that the calculated secondary chemical shifts under ff14IDPs force field are in quantitative agreement with the data of NMR experiment for five tested systems. In addition, the simulation results show that ff14IDPs can still be used to model structural proteins, such as tested lysozyme and ubiquitin, with better performance in coil regions than the original general Amber force field ff14SB. These findings confirm that the newly developed Amber ff14IDPs force field is a robust model for improving the conformation sampling of IDPs. PMID:27484738

Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

PubMed

Zhu, Shun; Travis, Sue M; Elcock, Adrian H

2013-07-09

A major current challenge for drug design efforts focused on protein kinases is the development of drug resistance caused by spontaneous mutations in the kinase catalytic domain. The ubiquity of this problem means that it would be advantageous to develop fast, effective computational methods that could be used to determine the effects of potential resistance-causing mutations before they arise in a clinical setting. With this long-term goal in mind, we have conducted a combined experimental and computational study of the thermodynamic effects of active-site mutations on a well-characterized and high-affinity interaction between a protein kinase and a small-molecule inhibitor. Specifically, we developed a fluorescence-based assay to measure the binding free energy of the small-molecule inhibitor, SB203580, to the p38α MAP kinase and used it measure the inhibitor's affinity for five different kinase mutants involving two residues (Val38 and Ala51) that contact the inhibitor in the crystal structure of the inhibitor-kinase complex. We then conducted long, explicit-solvent thermodynamic integration (TI) simulations in an attempt to reproduce the experimental relative binding affinities of the inhibitor for the five mutants; in total, a combined simulation time of 18.5 μs was obtained. Two widely used force fields - OPLS-AA/L and Amber ff99SB-ILDN - were tested in the TI simulations. Both force fields produced excellent agreement with experiment for three of the five mutants; simulations performed with the OPLS-AA/L force field, however, produced qualitatively incorrect results for the constructs that contained an A51V mutation. Interestingly, the discrepancies with the OPLS-AA/L force field could be rectified by the imposition of position restraints on the atoms of the protein backbone and the inhibitor without destroying the agreement for other mutations; the ability to reproduce experiment depended, however, upon the strength of the restraints' force constant. Imposition of position restraints in corresponding simulations that used the Amber ff99SB-ILDN force field had little effect on their ability to match experiment. Overall, the study shows that both force fields can work well for predicting the effects of active-site mutations on small molecule binding affinities and demonstrates how a direct combination of experiment and computation can be a powerful strategy for developing an understanding of protein-inhibitor interactions.

Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy.

PubMed

Rangl, Martina; Leitner, Michael; Riihimäki, Tiina; Lehtonen, Soili; Hytönen, Vesa P; Gruber, Hermann J; Kulomaa, Markku; Hinterdorfer, Peter; Ebner, Andreas

2014-02-01

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (k(off)) and energy landscape distances (x(β)) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the x(β) values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications. Copyright © 2014 John Wiley & Sons, Ltd.

Evidence of protein-free homology recognition in magnetic bead force–extension experiments

PubMed Central

(O’) Lee, D. J.; Danilowicz, C.; Rochester, C.; Prentiss, M.

2016-01-01

Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data. PMID:27493568

Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations.

PubMed

Velazquez, Hector A; Hamelberg, Donald

2015-02-21

Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Velazquez, Hector A.; Hamelberg, Donald

2015-02-01

Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

Curvature Forces in Membrane Lipid-Protein Interactions

PubMed Central

Brown, Michael F.

2012-01-01

Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes of membrane proteins—involving folding, stability, and membrane shape transitions—potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics, and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function. PMID:23163284

Single-Molecule Microscopy and Force Spectroscopy of Membrane Proteins

NASA Astrophysics Data System (ADS)

Engel, Andreas; Janovjak, Harald; Fotiadis, Dimtrios; Kedrov, Alexej; Cisneros, David; Müller, Daniel J.

Single-molecule atomic force microscopy (AFM) provides novel ways to characterize the structure-function relationship of native membrane proteins. High-resolution AFM topographs allow observing the structure of single proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. We will review these feasibilities illustrating examples of membrane proteins in native and reconstituted membranes. Classification of individual topographs of single proteins allows understanding the principles of motions of their extrinsic domains, to learn about their local structural flexibilities and to find the entropy minima of certain conformations. Combined with the visualization of functionally related conformational changes these insights allow understanding why certain flexibilities are required for the protein to function and how structurally flexible regions allow certain conformational changes. Complementary to AFM imaging, single-molecule force spectroscopy (SMFS) experiments detect molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to measure interactions that stabilize secondary structures such as transmembrane α-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the locations of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes.

The Force Exerted by the Membrane Potential During Protein Import into the Mitochondrial Matrix

NASA Technical Reports Server (NTRS)

Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

2002-01-01

The electrostatic force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated and found to vary from 1.4 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 12 to 6.5 Angstroms, its measured range. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a nonaqueous pore gives a force of 3.1 pN per unit charge, which represents an upper limit. When applied to mitochondrial import experiments on the protein harness, these results imply that a force of 11 plus or minus 4 pN is sufficient to catalyze the unfolding of harness during import. Comparison of these results with unfolding forces measured using atomic force microscopy suggests that the two are not inconsistent.

The influence of disulfide bonds on the mechanical stability of proteins is context dependent.

PubMed

Manteca, Aitor; Alonso-Caballero, Álvaro; Fertin, Marie; Poly, Simon; De Sancho, David; Perez-Jimenez, Raul

2017-08-11

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. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Motor-motor interactions in ensembles of muscle myosin: using theory to connect single molecule to ensemble measurements

NASA Astrophysics Data System (ADS)

Walcott, Sam

2013-03-01

Interactions between the proteins actin and myosin drive muscle contraction. Properties of a single myosin interacting with an actin filament are largely known, but a trillion myosins work together in muscle. We are interested in how single-molecule properties relate to ensemble function. Myosin's reaction rates depend on force, so ensemble models keep track of both molecular state and force on each molecule. These models make subtle predictions, e.g. that myosin, when part of an ensemble, moves actin faster than when isolated. This acceleration arises because forces between molecules speed reaction kinetics. Experiments support this prediction and allow parameter estimates. A model based on this analysis describes experiments from single molecule to ensemble. In vivo, actin is regulated by proteins that, when present, cause the binding of one myosin to speed the binding of its neighbors; binding becomes cooperative. Although such interactions preclude the mean field approximation, a set of linear ODEs describes these ensembles under simplified experimental conditions. In these experiments cooperativity is strong, with the binding of one molecule affecting ten neighbors on either side. We progress toward a description of myosin ensembles under physiological conditions.

Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field.

PubMed

Xu, Dong; Zhang, Yang

2012-07-01

Ab initio protein folding is one of the major unsolved problems in computational biology owing to the difficulties in force field design and conformational search. We developed a novel program, QUARK, for template-free protein structure prediction. Query sequences are first broken into fragments of 1-20 residues where multiple fragment structures are retrieved at each position from unrelated experimental structures. Full-length structure models are then assembled from fragments using replica-exchange Monte Carlo simulations, which are guided by a composite knowledge-based force field. A number of novel energy terms and Monte Carlo movements are introduced and the particular contributions to enhancing the efficiency of both force field and search engine are analyzed in detail. QUARK prediction procedure is depicted and tested on the structure modeling of 145 nonhomologous proteins. Although no global templates are used and all fragments from experimental structures with template modeling score >0.5 are excluded, QUARK can successfully construct 3D models of correct folds in one-third cases of short proteins up to 100 residues. In the ninth community-wide Critical Assessment of protein Structure Prediction experiment, QUARK server outperformed the second and third best servers by 18 and 47% based on the cumulative Z-score of global distance test-total scores in the FM category. Although ab initio protein folding remains a significant challenge, these data demonstrate new progress toward the solution of the most important problem in the field. Copyright © 2012 Wiley Periodicals, Inc.

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

PubMed

Ott, Wolfgang; Jobst, Markus A; Bauer, Magnus S; Durner, Ellis; Milles, Lukas F; Nash, Michael A; Gaub, Hermann E

2017-06-27

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

Generalized rules for the optimization of elastic network models

NASA Astrophysics Data System (ADS)

Lezon, Timothy; Eyal, Eran; Bahar, Ivet

2009-03-01

Elastic network models (ENMs) are widely employed for approximating the coarse-grained equilibrium dynamics of proteins using only a few parameters. An area of current focus is improving the predictive accuracy of ENMs by fine-tuning their force constants to fit specific systems. Here we introduce a set of general rules for assigning ENM force constants to residue pairs. Using a novel method, we construct ENMs that optimally reproduce experimental residue covariances from NMR models of 68 proteins. We analyze the optimal interactions in terms of amino acid types, pair distances and local protein structures to identify key factors in determining the effective spring constants. When applied to several unrelated globular proteins, our method shows an improved correlation with experiment over a standard ENM. We discuss the physical interpretation of our findings as well as its implications in the fields of protein folding and dynamics.

Nanomechanics of Protein Unfolding outside Protease Nanopores

NASA Astrophysics Data System (ADS)

Luan, Binquan; Zhou, Ruhong

Protein folding and unfolding have been the subject of active research for decades. Most of previous studies in protein unfolding were focused on temperature, chemical and/or force (such as in AFM) induced denaturations. Recent studies on the functional roles of proteasomes (such as ClpXP) revealed a novel unfolding process in cell, during which a target protein is mechanically unfolded and pulled into a confined, pore-like geometry for degradation. While the proteasome nanomachine has been extensively studied, the mechanism for unfolding proteins with the proteasome pore is still poorly understood. Here, we investigate the mechanical unfolding process of ubiquitin with (or really outside) an idealized proteasome pore, and compare such process with that in the AFM pulling experiment. Unexpectedly, the required force by a proteosome can be much smaller than that by the AFM. Simulation results also unveiled different nanomechanics, tearing fracture vs. shearing friction, in these two distinct types of mechanical unfoldings.

Physical driving force of actomyosin motility based on the hydration effect.

PubMed

Suzuki, Makoto; Mogami, George; Ohsugi, Hideyuki; Watanabe, Takahiro; Matubayasi, Nobuyuki

2017-12-01

We propose a driving force hypothesis based on previous thermodynamics, kinetics and structural data as well as additional experiments and calculations presented here on water-related phenomena in the actomyosin systems. Although Szent-Györgyi pointed out the importance of water in muscle contraction in 1951, few studies have focused on the water science of muscle because of the difficulty of analyzing hydration properties of the muscle proteins, actin, and myosin. The thermodynamics and energetics of muscle contraction are linked to the water-mediated regulation of protein-ligand and protein-protein interactions along with structural changes in protein molecules. In this study, we assume the following two points: (1) the periodic electric field distribution along an actin filament (F-actin) is unidirectionally modified upon binding of myosin subfragment 1 (M or myosin S1) with ADP and inorganic phosphate Pi (M.ADP.Pi complex) and (2) the solvation free energy of myosin S1 depends on the external electric field strength and the solvation free energy of myosin S1 in close proximity to F-actin can become the potential force to drive myosin S1 along F-actin. The first assumption is supported by integration of experimental reports. The second assumption is supported by model calculations utilizing molecular dynamics (MD) simulation to determine solvation free energies of a small organic molecule and two small proteins. MD simulations utilize the energy representation method (ER) and the roughly proportional relationship between the solvation free energy and the solvent-accessible surface area (SASA) of the protein. The estimated driving force acting on myosin S1 is as high as several piconewtons (pN), which is consistent with the experimentally observed force. © 2017 Wiley Periodicals, Inc.

Pathways and intermediates in forced unfolding of spectrin repeats.

PubMed

Altmann, Stephan M; Grünberg, Raik G; Lenne, Pierre-François; Ylänne, Jari; Raae, Arnt; Herbert, Kristina; Saraste, Matti; Nilges, Michael; Hörber, J K Heinrich

2002-08-01

Spectrin repeats are triple-helical coiled-coil domains found in many proteins that are regularly subjected to mechanical stress. We used atomic force microscopy technique and steered molecular dynamics simulations to study the behavior of a wild-type spectrin repeat and two mutants. The experiments indicate that spectrin repeats can form stable unfolding intermediates when subjected to external forces. In the simulations the unfolding proceeded via a variety of pathways. Stable intermediates were associated to kinking of the central helix close to a proline residue. A mutant stabilizing the central helix showed no intermediates in experiments, in agreement with simulation. Spectrin repeats may thus function as elastic elements, extendable to intermediate states at various lengths.

Rheology of biological macromolecules

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila Dinesh

Proteins have interesting mechanical properties in addition to the remarkable functionality. For example, Guanylate kinase is an enzyme that catalyzes Guano- sine monophosphate (GMP) to Guanosine diphosphate (GDP) conversion and this enzyme is approximately 5 nm in size. A gold nano particle of similar size shows linear elasticity for strains up to ˜ 0.1% and shows plastic deformation beyond that, whereas the enzyme Guanylate kinase can have strains up to 1 % with reversible deformation. Our experiments show many different regimes of the mechanical response before the plastic deformation of these proteins. In this dissertation, I study the materials properties of two classes of proteins, an ion channel protein and a transferase, which is a globular protein. The experimental techniques to study the materials properties of these proteins were uniquely developed at the Zocchi lab. Therefore, we were able to observe previously unknown characteristics of these folded proteins. The mechanical properties of the voltage gated potassium channel KvAP was studied by applying AC depolarizing voltages. This technique gave new information about the system that was not seen in the previous studies. These previous experiments were based on applying DC depolarizing voltage steps across the membrane to study the ionic current. By monitoring the ionic current at different depolarizing voltage steps, the DC gating process of the channel could be under- stood. We probed the channel using AC depolarizing signals instead of DC pulses and the ionic current revealed new behaviors, which cannot be predicted with the DC response. We found that the conformational motion of the voltage sensing domain of the ion channel shows internal dissipation. Further, a new non linearity in the dissipation parameter was found in which the dissipation parameter increased with the shear rate of the applied force. Previous studies at the Zocchi lab used a nano rheology experiment on the protein Guanylate kinase to study the mechanical properties of a globular protein. The protein was subjected to a mechanical force and the deformation was measured with sub-Angstrom resolution. We found that the protein shows a linear elasticity regime for low forcing and viscoelastic behavior for high forcing. The internal viscosity of the protein is due to the internal dissipation of the protein. This dissertation takes the work on nano rheology of proteins further by studying the temperature effect on the materials properties of the protein and the contribution of the surface of the protein to the observed mechanics. In addition to studying the materials properties of proteins, we used proteins to design new biomimetic systems. The first system covered in this dissertation is the development of a novel sensor platform for molecules. In this sensor, we detect the change in the stiffness of the substrate upon binding a target rather than the usual scheme of detecting the change in mass upon binding of a target. By combining the nano rheology setup with localized surface plasmon resonance, this sensor platform yields a very robust signal. The other biomimetic system that is discussed here is an artificial axon is constructed with ion channels and lipid bilayers.

Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy.

PubMed

Baumann, Fabian; Bauer, Magnus S; Milles, Lukas F; Alexandrovich, Alexander; Gaub, Hermann E; Pippig, Diana A

2016-01-01

Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.

Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy

NASA Astrophysics Data System (ADS)

Baumann, Fabian; Bauer, Magnus S.; Milles, Lukas F.; Alexandrovich, Alexander; Gaub, Hermann E.; Pippig, Diana A.

2016-01-01

Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.

Kinesin and Dynein Mechanics: Measurement Methods and Research Applications.

PubMed

Abraham, Zachary; Hawley, Emma; Hayosh, Daniel; Webster-Wood, Victoria A; Akkus, Ozan

2018-02-01

Motor proteins play critical roles in the normal function of cells and proper development of organisms. Among motor proteins, failings in the normal function of two types of proteins, kinesin and dynein, have been shown to lead many pathologies, including neurodegenerative diseases and cancers. As such, it is critical to researchers to understand the underlying mechanics and behaviors of these proteins, not only to shed light on how failures may lead to disease, but also to guide research toward novel treatment and nano-engineering solutions. To this end, many experimental techniques have been developed to measure the force and motility capabilities of these proteins. This review will (a) discuss such techniques, specifically microscopy, atomic force microscopy (AFM), optical trapping, and magnetic tweezers, and (b) the resulting nanomechanical properties of motor protein functions such as stalling force, velocity, and dependence on adenosine triphosophate (ATP) concentrations will be comparatively discussed. Additionally, this review will highlight the clinical importance of these proteins. Furthermore, as the understanding of the structure and function of motor proteins improves, novel applications are emerging in the field. Specifically, researchers have begun to modify the structure of existing proteins, thereby engineering novel elements to alter and improve native motor protein function, or even allow the motor proteins to perform entirely new tasks as parts of nanomachines. Kinesin and dynein are vital elements for the proper function of cells. While many exciting experiments have shed light on their function, mechanics, and applications, additional research is needed to completely understand their behavior.

An open source/real-time atomic force microscope architecture to perform customizable force spectroscopy experiments.

PubMed

Materassi, Donatello; Baschieri, Paolo; Tiribilli, Bruno; Zuccheri, Giampaolo; Samorì, Bruno

2009-08-01

We describe the realization of an atomic force microscope architecture designed to perform customizable experiments in a flexible and automatic way. Novel technological contributions are given by the software implementation platform (RTAI-LINUX), which is free and open source, and from a functional point of view, by the implementation of hard real-time control algorithms. Some other technical solutions such as a new way to estimate the optical lever constant are described as well. The adoption of this architecture provides many degrees of freedom in the device behavior and, furthermore, allows one to obtain a flexible experimental instrument at a relatively low cost. In particular, we show how such a system has been employed to obtain measures in sophisticated single-molecule force spectroscopy experiments [Fernandez and Li, Science 303, 1674 (2004)]. Experimental results on proteins already studied using the same methodologies are provided in order to show the reliability of the measure system.

A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations.

PubMed

Duan, Yong; Wu, Chun; Chowdhury, Shibasish; Lee, Mathew C; Xiong, Guoming; Zhang, Wei; Yang, Rong; Cieplak, Piotr; Luo, Ray; Lee, Taisung; Caldwell, James; Wang, Junmei; Kollman, Peter

2003-12-01

Molecular mechanics models have been applied extensively to study the dynamics of proteins and nucleic acids. Here we report the development of a third-generation point-charge all-atom force field for proteins. Following the earlier approach of Cornell et al., the charge set was obtained by fitting to the electrostatic potentials of dipeptides calculated using B3LYP/cc-pVTZ//HF/6-31G** quantum mechanical methods. The main-chain torsion parameters were obtained by fitting to the energy profiles of Ace-Ala-Nme and Ace-Gly-Nme di-peptides calculated using MP2/cc-pVTZ//HF/6-31G** quantum mechanical methods. All other parameters were taken from the existing AMBER data base. The major departure from previous force fields is that all quantum mechanical calculations were done in the condensed phase with continuum solvent models and an effective dielectric constant of epsilon = 4. We anticipate that this force field parameter set will address certain critical short comings of previous force fields in condensed-phase simulations of proteins. Initial tests on peptides demonstrated a high-degree of similarity between the calculated and the statistically measured Ramanchandran maps for both Ace-Gly-Nme and Ace-Ala-Nme di-peptides. Some highlights of our results include (1) well-preserved balance between the extended and helical region distributions, and (2) favorable type-II poly-proline helical region in agreement with recent experiments. Backward compatibility between the new and Cornell et al. charge sets, as judged by overall agreement between dipole moments, allows a smooth transition to the new force field in the area of ligand-binding calculations. Test simulations on a large set of proteins are also discussed. Copyright 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1999-2012, 2003

Unraveling protein-protein interactions in clathrin assemblies via atomic force spectroscopy.

PubMed

Jin, Albert J; Lafer, Eileen M; Peng, Jennifer Q; Smith, Paul D; Nossal, Ralph

2013-03-01

Atomic force microscopy (AFM), single molecule force spectroscopy (SMFS), and single particle force spectroscopy (SPFS) are used to characterize intermolecular interactions and domain structures of clathrin triskelia and clathrin-coated vesicles (CCVs). The latter are involved in receptor-mediated endocytosis (RME) and other trafficking pathways. Here, we subject individual triskelia, bovine-brain CCVs, and reconstituted clathrin-AP180 coats to AFM-SMFS and AFM-SPFS pulling experiments and apply novel analytics to extract force-extension relations from very large data sets. The spectroscopic fingerprints of these samples differ markedly, providing important new information about the mechanism of CCV uncoating. For individual triskelia, SMFS reveals a series of events associated with heavy chain alpha-helix hairpin unfolding, as well as cooperative unraveling of several hairpin domains. SPFS of clathrin assemblies exposes weaker clathrin-clathrin interactions that are indicative of inter-leg association essential for RME and intracellular trafficking. Clathrin-AP180 coats are energetically easier to unravel than the coats of CCVs, with a non-trivial dependence on force-loading rate. Published by Elsevier Inc.

Swelling and Softening of the Cowpea Chlorotic Mottle Virus in Response to pH Shifts

PubMed Central

Wilts, Bodo D.; Schaap, Iwan A.T.; Schmidt, Christoph F.

2015-01-01

Cowpea chlorotic mottle virus (CCMV) forms highly elastic icosahedral protein capsids that undergo a characteristic swelling transition when the pH is raised from 5 to 7. Here, we performed nano-indentation experiments using an atomic force microscope to track capsid swelling and measure the shells’ Young’s modulus at the same time. When we chelated Ca2+ ions and raised the pH, we observed a gradual swelling of the RNA-filled capsids accompanied by a softening of the shell. Control experiments with empty wild-type virus and a salt-stable mutant revealed that the softening was not strictly coupled to the swelling of the protein shells. Our data suggest that a pH increase and Ca2+ chelation lead primarily to a loosening of contacts within the protein shell, resulting in a softening of the capsid. This appears to render the shell metastable and make swelling possible when repulsive forces among the capsid proteins become large enough, which is known to be followed by capsid disassembly at even higher pH. Thus, softening and swelling are likely to play a role during inoculation. PMID:25992732

Ab Initio Protein Structure Assembly Using Continuous Structure Fragments and Optimized Knowledge-based Force Field

PubMed Central

Xu, Dong; Zhang, Yang

2012-01-01

Ab initio protein folding is one of the major unsolved problems in computational biology due to the difficulties in force field design and conformational search. We developed a novel program, QUARK, for template-free protein structure prediction. Query sequences are first broken into fragments of 1–20 residues where multiple fragment structures are retrieved at each position from unrelated experimental structures. Full-length structure models are then assembled from fragments using replica-exchange Monte Carlo simulations, which are guided by a composite knowledge-based force field. A number of novel energy terms and Monte Carlo movements are introduced and the particular contributions to enhancing the efficiency of both force field and search engine are analyzed in detail. QUARK prediction procedure is depicted and tested on the structure modeling of 145 non-homologous proteins. Although no global templates are used and all fragments from experimental structures with template modeling score (TM-score) >0.5 are excluded, QUARK can successfully construct 3D models of correct folds in 1/3 cases of short proteins up to 100 residues. In the ninth community-wide Critical Assessment of protein Structure Prediction (CASP9) experiment, QUARK server outperformed the second and third best servers by 18% and 47% based on the cumulative Z-score of global distance test-total (GDT-TS) scores in the free modeling (FM) category. Although ab initio protein folding remains a significant challenge, these data demonstrate new progress towards the solution of the most important problem in the field. PMID:22411565

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

DOE PAGES

Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; ...

2016-07-27

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different beta-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin alpha-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels aremore » consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. In conclusion, the obtained results demonstrate that the general concept of "polymers pushing polymers" is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores.« less

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

PubMed Central

Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; Gurnev, Philip A.; Muthukumar, Murugappan; Parsegian, V. Adrian

2016-01-01

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different β-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of “polymers pushing polymers” is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores. PMID:27466408

Actin stress in cell reprogramming

PubMed Central

Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

2014-01-01

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

Single Molecule Study of DNA Organization and Recombination

NASA Astrophysics Data System (ADS)

Xiao, Botao

We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force, which depends on salt concentration. We also report experiments with the recombinase Hin mutant H107Y. The synapse formation is demonstrated with single DNA containing two hix sites. We further show preliminary data for cleavage and subunit rotation from a braiding assay. These direct observations elucidate the recombination mechanism.

Thermal denaturing of mutant lysozyme with both the OPLSAA and the CHARMM force fields.

PubMed

Eleftheriou, Maria; Germain, Robert S; Royyuru, Ajay K; Zhou, Ruhong

2006-10-18

Biomolecular simulations enabled by massively parallel supercomputers such as BlueGene/L promise to bridge the gap between the currently accessible simulation time scale and the experimental time scale for many important protein folding processes. In this study, molecular dynamics simulations were carried out for both the wild-type and the mutant hen lysozyme (TRP62GLY) to study the single mutation effect on lysozyme stability and misfolding. Our thermal denaturing simulations at 400-500 K with both the OPLSAA and the CHARMM force fields show that the mutant structure is indeed much less stable than the wild-type, which is consistent with the recent urea denaturing experiment (Dobson et al. Science 2002, 295, 1719-1722; Nature 2003, 424, 783-788). Detailed results also reveal that the single mutation TRP62GLY first induces the loss of native contacts in the beta-domain region of the lysozyme protein at high temperatures, and then the unfolding process spreads into the alpha-domain region through Helix C. Even though the OPLSAA force field in general shows a more stable protein structure than does the CHARMM force field at high temperatures, the two force fields examined here display qualitatively similar results for the misfolding process, indicating that the thermal denaturing of the single mutation is robust and reproducible with various modern force fields.

A Discontinuous Potential Model for Protein-Protein Interactions.

PubMed

Shao, Qing; Hall, Carol K

2016-01-01

Protein-protein interactions play an important role in many biologic and industrial processes. In this work, we develop a two-bead-per-residue model that enables us to account for protein-protein interactions in a multi-protein system using discontinuous molecular dynamics simulations. This model deploys discontinuous potentials to describe the non-bonded interactions and virtual bonds to keep proteins in their native state. The geometric and energetic parameters are derived from the potentials of mean force between sidechain-sidechain, sidechain-backbone, and backbone-backbone pairs. The energetic parameters are scaled with the aim of matching the second virial coefficient of lysozyme reported in experiment. We also investigate the performance of several bond-building strategies.

A simple polymeric model describes cell nuclear mechanical response

NASA Astrophysics Data System (ADS)

Banigan, Edward; Stephens, Andrew; Marko, John

The cell nucleus must continually resist inter- and intracellular mechanical forces, and proper mechanical response is essential to basic cell biological functions as diverse as migration, differentiation, and gene regulation. Experiments probing nuclear mechanics reveal that the nucleus stiffens under strain, leading to two characteristic regimes of force response. This behavior depends sensitively on the intermediate filament protein lamin A, which comprises the outer layer of the nucleus, and the properties of the chromatin interior. To understand these mechanics, we study a simulation model of a polymeric shell encapsulating a semiflexible polymer. This minimalistic model qualitatively captures the typical experimental nuclear force-extension relation and observed nuclear morphologies. Using a Flory-like theory, we explain the simulation results and mathematically estimate the force-extension relation. The model and experiments suggest that chromatin organization is a dominant contributor to nuclear mechanics, while the lamina protects cell nuclei from large deformations.

Actin growth profile in clathrin-mediated endocytosis

NASA Astrophysics Data System (ADS)

Tweten, D. J.; Bayly, P. V.; Carlsson, A. E.

2017-05-01

Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000 -10 000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.

Tug of war in motility assay experiments

NASA Astrophysics Data System (ADS)

Hexner, Daniel; Kafri, Yariv

2009-09-01

The dynamics of two groups of molecular motors pulling in opposite directions on a rigid filament is studied theoretically. To this end we first consider the behavior of one set of motors pulling in a single direction against an external force using a new mean-field approach. Based on these results we analyze a similar setup with two sets of motors pulling in opposite directions in a tug of war in the presence of an external force. In both cases we find that the interplay of fluid friction and protein friction leads to a complex phase diagram where the force-velocity relations can exhibit regions of bistability and spontaneous symmetry breaking. Finally, motivated by recent work, we turn to the case of motility assay experiments where motors bound to a surface push on a bundle of filaments. We find that, depending on the absence or the presence of bistability in the force-velocity curve at zero force, the bundle exhibits anomalous or biased diffusion on long-time and large-length scales.

On the function of chitin synthase extracellular domains in biomineralization.

PubMed

Weiss, Ingrid M; Lüke, Florian; Eichner, Norbert; Guth, Christina; Clausen-Schaumann, Hauke

2013-08-01

Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface. Copyright © 2013 Elsevier Inc. All rights reserved.

Design principles for robust vesiculation in clathrin-mediated endocytosis

PubMed Central

Hassinger, Julian E.; Oster, George; Drubin, David G.; Rangamani, Padmini

2017-01-01

A critical step in cellular-trafficking pathways is the budding of membranes by protein coats, which recent experiments have demonstrated can be inhibited by elevated membrane tension. The robustness of processes like clathrin-mediated endocytosis (CME) across a diverse range of organisms and mechanical environments suggests that the protein machinery in this process has evolved to take advantage of some set of physical design principles to ensure robust vesiculation against opposing forces like membrane tension. Using a theoretical model for membrane mechanics and membrane protein interaction, we have systematically investigated the influence of membrane rigidity, curvature induced by the protein coat, area covered by the protein coat, membrane tension, and force from actin polymerization on bud formation. Under low tension, the membrane smoothly evolves from a flat to budded morphology as the coat area or spontaneous curvature increases, whereas the membrane remains essentially flat at high tensions. At intermediate, physiologically relevant, tensions, the membrane undergoes a “snap-through instability” in which small changes in the coat area, spontaneous curvature or membrane tension cause the membrane to “snap” from an open, U-shape to a closed bud. This instability can be smoothed out by increasing the bending rigidity of the coat, allowing for successful budding at higher membrane tensions. Additionally, applied force from actin polymerization can bypass the instability by inducing a smooth transition from an open to a closed bud. Finally, a combination of increased coat rigidity and force from actin polymerization enables robust vesiculation even at high membrane tensions. PMID:28126722

Force generation by titin folding.

PubMed

Mártonfalvi, Zsolt; Bianco, Pasquale; Naftz, Katalin; Ferenczy, György G; Kellermayer, Miklós

2017-07-01

Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (<10 pN), extension fluctuated without resolvable discrete events. In position-clamp experiments, the time-dependent force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles. © 2017 The Protein Society.

The film tells the story: Physical-chemical characteristics of IgG at the liquid-air interface.

PubMed

Koepf, Ellen; Schroeder, Rudolf; Brezesinski, Gerald; Friess, Wolfgang

2017-10-01

The presence of liquid-air interfaces in protein pharmaceuticals is known to negatively impact product stability. Nevertheless, the mechanisms behind interface-related protein aggregation are not yet fully understood. Little is known about the physical-chemical behavior of proteins adsorbed to the interface. Therefore, the combinatorial use of appropriate surface-sensitive analytical methods such as Langmuir trough experiments, Infrared Reflection-Absorption Spectroscopy (IRRAS), Brewster Angle Microscopy (BAM), and Atomic Force Microscopy (AFM) is highly expedient to uncover structures and events at the liquid-air interface directly. Concentration-dependent adsorption of a human immunoglobulin G (IgG) and characteristic surface-pressure/area isotherms substantiated the amphiphilic nature of the protein molecules as well as the formation of a compressible protein film at the liquid-air interface. Upon compression, the IgG molecules do not readily desorb but form a highly compressible interfacial film. IRRA spectra proved not only the presence of the protein at the interface, but also showed that the secondary structure does not change considerably during adsorption or compression. IRRAS experiments at different angles of incidence indicated that the film thickness and/or packing density increases upon compression. Furthermore, BAM images exposed the presence of a coherent but heterogeneous distribution of the protein at the interface. Topographical differences within the protein film after adsorption, compression and decompression were revealed using underwater AFM. The combinatorial use of physical-chemical, spectroscopic and microscopic methods provided useful insights into the liquid-air interfacial protein behavior and revealed the formation of a continuous but inhomogeneous film of native-like protein molecules whose topographical appearance is affected by compressive forces. Copyright © 2017 Elsevier B.V. All rights reserved.

Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on Surfaces

NASA Astrophysics Data System (ADS)

Christenson, Wayne B.

Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is alpha Mbeta2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights.

Stretching of Single Polymer Chains Using the Atomic Force Microscope

NASA Astrophysics Data System (ADS)

Ortiz, C.; van der Vegte, E. W.; van Swieten, E.; Robillard, G. T.; Hadziioannou, G.

1998-03-01

A variety of macroscopic phenomenon involve "nanoscale" polymer deformation including rubber elasticity, shear yielding, strain hardening, stress relaxation, fracture, and flow. With the advent of new and improved experimental techniques, such as the atomic force microscope (AFM), the probing of physical properties of polymers has reached finer and finer scales. The development of mixed self-assembling monolayer techniques and the chemical functionalization of AFM probe tips has allowed for mechanical experiments on single polymer chains of molecular dimensions. In our experiments, mixed monolayers are prepared in which end-functionalized, flexible polymer chains of thiol-terminated poly(methacrylic acid) are covalently bonded, isolated, and randomly distributed on gold substrates. The coils are then imaged, tethered to a gold-coated AFM tip, and stretched between the tip and the substrate in a conventional force / distance experiment. An increase in the attractive force due to entropic, elastic resistance to stretching, as well as fracture of the polymer chain is observed. The effect of chain stiffness, topological constraints, strain rate, mechanical hysteresis, and stress relaxation were investigated. Force modulation techniques were also employed in order to image the viscoelastic character of the polymer chains. Parallel work includes similar studies of biological systems such as wheat gluten proteins and polypeptides.

The roles of fluid motion and other transport phenomena in the morphology of materials

NASA Technical Reports Server (NTRS)

Saville, D. A.

1993-01-01

Two crystallization problems were studied: the growth of protein crystals, in particular the influence of colloidal forces and convection, and the influence of interface resistance on the growth of dendritic crystals. The protein study involved both experimental and theoretical work; the work of dendrites was entirely theoretical. In the study of protein crystallization, experiments were carried out where crystals were grown in the presence and absence of natural convection. No evidence was found that convection retards crystal growth. The theoretical study focused on the influence of colloidal forces (electrostatic and London-van der Waals) on the interaction between a protein molecule and a flat crystal surface. It was shown that the interaction is extremely sensitive to colloidal forces and that electrostatic interactions play a strong role in deciding whether or not a molecule will find a favorable site for adsorption. In the study of dendritic growth, the role of an interfacial resistance on the selection processes was examined. Using a computational scheme, it was found that the selected velocity is strongly dependent on the magnitude of the interfacial resistance to heat transfer. This is a possible explanation for discrepancies between the theoretical and experimental results on succinonitrile.

The roles of fluid motion and other transport phenomena in the morphology of materials

NASA Astrophysics Data System (ADS)

Saville, D. A.

1993-11-01

Two crystallization problems were studied: the growth of protein crystals, in particular the influence of colloidal forces and convection, and the influence of interface resistance on the growth of dendritic crystals. The protein study involved both experimental and theoretical work; the work of dendrites was entirely theoretical. In the study of protein crystallization, experiments were carried out where crystals were grown in the presence and absence of natural convection. No evidence was found that convection retards crystal growth. The theoretical study focused on the influence of colloidal forces (electrostatic and London-van der Waals) on the interaction between a protein molecule and a flat crystal surface. It was shown that the interaction is extremely sensitive to colloidal forces and that electrostatic interactions play a strong role in deciding whether or not a molecule will find a favorable site for adsorption. In the study of dendritic growth, the role of an interfacial resistance on the selection processes was examined. Using a computational scheme, it was found that the selected velocity is strongly dependent on the magnitude of the interfacial resistance to heat transfer. This is a possible explanation for discrepancies between the theoretical and experimental results on succinonitrile.

Sampling Long- versus Short-Range Interactions Defines the Ability of Force Fields To Reproduce the Dynamics of Intrinsically Disordered Proteins.

PubMed

Mercadante, Davide; Wagner, Johannes A; Aramburu, Iker V; Lemke, Edward A; Gräter, Frauke

2017-09-12

Molecular dynamics (MD) simulations have valuably complemented experiments describing the dynamics of intrinsically disordered proteins (IDPs), particularly since the proposal of models to solve the artificial collapse of IDPs in silico. Such models suggest redefining nonbonded interactions, by either increasing water dispersion forces or adopting the Kirkwood-Buff force field. These approaches yield extended conformers that better comply with experiments, but it is unclear if they all sample the same intrachain dynamics of IDPs. We have tested this by employing MD simulations and single-molecule Förster resonance energy transfer spectroscopy to sample the dimensions of systems with different sequence compositions, namely strong and weak polyelectrolytes. For strong polyelectrolytes in which charge effects dominate, all the proposed solutions equally reproduce the expected ensemble's dimensions. For weak polyelectrolytes, at lower cutoffs, force fields abnormally alter intrachain dynamics, overestimating excluded volume over chain flexibility or reporting no difference between the dynamics of different chains. The TIP4PD water model alone can reproduce experimentally observed changes in extensions (dimensions), but not quantitatively and with only weak statistical significance. Force field limitations are reversed with increased interaction cutoffs, showing that chain dynamics are critically defined by the presence of long-range interactions. Force field analysis aside, our study provides the first insights into how long-range interactions critically define IDP dimensions and raises the question of which length range is crucial to correctly sample the overall dimensions and internal dynamics of the large group of weakly charged yet highly polar IDPs.

Unfolding of Proteins: Thermal and Mechanical Unfolding

NASA Technical Reports Server (NTRS)

Hur, Joe S.; Darve, Eric

2004-01-01

We have employed a Hamiltonian model based on a self-consistent Gaussian appoximation to examine the unfolding process of proteins in external - both mechanical and thermal - force elds. The motivation was to investigate the unfolding pathways of proteins by including only the essence of the important interactions of the native-state topology. Furthermore, if such a model can indeed correctly predict the physics of protein unfolding, it can complement more computationally expensive simulations and theoretical work. The self-consistent Gaussian approximation by Micheletti et al. has been incorporated in our model to make the model mathematically tractable by signi cantly reducing the computational cost. All thermodynamic properties and pair contact probabilities are calculated by simply evaluating the values of a series of Incomplete Gamma functions in an iterative manner. We have compared our results to previous molecular dynamics simulation and experimental data for the mechanical unfolding of the giant muscle protein Titin (1TIT). Our model, especially in light of its simplicity and excellent agreement with experiment and simulation, demonstrates the basic physical elements necessary to capture the mechanism of protein unfolding in an external force field.

Single-molecule experiments in biological physics: methods and applications.

PubMed

Ritort, F

2006-08-16

I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.

TOPICAL REVIEW: Single-molecule experiments in biological physics: methods and applications

NASA Astrophysics Data System (ADS)

Ritort, F.

2006-08-01

I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.

Alterations in the muscle force transfer apparatus in aged rats during unloading and reloading: Impact of microRNA-31.

PubMed

Hughes, David C; Marcotte, George R; Baehr, Leslie M; West, Daniel W D; Marshall, Andrea G; Ebert, Scott M; Davidyan, Arik; Adams, Christopher M; Bodine, Sue C; Baar, Keith

2018-05-03

Force transfer is integral for maintaining skeletal muscle structure and function. One important component is dystrophin. There is limited understanding of how force transfer is impacted by age and loading. Here, we investigate the force transfer apparatus in muscles of adult and old rats exposed to periods of disuse and reloading. Our results demonstrate an increase in dystrophin protein during the reloading phase in the adult TA muscle that is delayed in old. The consequence of this delay is an increased susceptibility towards contraction-induced muscle injury. Central to the lack of dystrophin protein is an increase in miR-31, a microRNA that inhibits dystrophin translation. In vivo electroporation with a miR-31 sponge led to increased dystrophin protein and decreased contraction-induced muscle injury in old skeletal muscle. Overall, our results detail the importance of the force transfer apparatus and provide new mechanisms for contraction-induced injury in aging skeletal muscle. In healthy muscle, the dystrophin-associated glycoprotein (DGC) and integrin/focal adhesion complexes, intermediate filaments, and Z-line proteins transmit force from the contractile proteins to the extracellular matrix. How loading and age affect these proteins is poorly understood. The experiments reported here sought to determine the effect of aging on the force transfer apparatus following muscle unloading and reloading. Adult (9 months) and old (29 months) rats were subjected to 14 days hindlimb unloading (HU) and 1, 3, 7 and 14 days of reloading (REL). The DGC complex, intermediate filament and z-line protein and mRNA levels, as well as dystrophin-targeting miRNAs (miR-31, -146b and -374) were examined in the tibialis anterior (TA) and medial gastrocnemius (MG) muscles at both ages. There was a significant increase in dystrophin protein levels (2.79-fold) upon 3 days of reloading in the adult TA muscle that did not occur in the old rats (p ≤ 0.05), and the rise in dystrophin protein occurred independent of dystrophin mRNA. The disconnect between dystrophin protein and mRNA levels can partially be explained age-dependent differences in miR-31. The impaired dystrophin response in aged muscle was followed by an increase in other force transfer proteins (β-dystroglycan, desmuslin and LIM) that was not sufficient to prevent membrane disruption and muscle injury early in the reloading period. Inserting a miR-31 sponge increased dystrophin protein and decreased contraction-induced injury in the TA (p ≤ 0.05). Collectively, these data suggest that increased miR-31 with age contributes to an impaired dystrophin response and increased muscle injury after disuse. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field.

PubMed

Freedman, Kevin J; Haq, S Raza; Edel, Joshua B; Jemth, Per; Kim, Min Jun

2013-01-01

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 10(6) V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.

SAW based micro- and acousto-fluidics in biomedicine

NASA Astrophysics Data System (ADS)

Ramasamy, Mouli; Varadan, Vijay K.

2017-04-01

Protein association starts with random collisions of individual proteins. Multiple collisions and rotational diffusion brings the molecules to a state of orientation. Majority of the protein associations are influenced by electrostatic interactions. To introduce: electrostatic rate enhancement, Brownian dynamics and transient complex theory has been traditionally used. Due to the recent advances in interdisciplinary sciences, an array of molecular assembly methods is being studied. Protein nanostructural assembly and macromolecular crowding are derived from the subsets of biochemistry to study protein-protein interactions and protein self-assembly. This paper tries to investigate the issue of enhancing the protein self-association rate, and bridging the gap between the simulations and experimental results. The methods proposed here include: electrostatic rate enhancement, macromolecular crowing, nanostructural protein assembly, microfluidics based approaches and magnetic force based approaches. Despite the suggestions of several methods, microfluidic and magnetic force based approaches seem to serve the need of protein assembly in a wider scale. Congruence of these approaches may also yield better results. Even though, these methods prove to be conceptually strong, to prevent the disagreement of theory and practice, a wide range of experiments is required. This proposal intends to study theoretical and experimental methods to successfully implement the aforementioned assembly strategies, and conclude with an extensive analysis of experimental data to address practical feasibility.

β-connectin studies by small-angle x-ray scattering and single-molecule force spectroscopy by atomic force microscopy

NASA Astrophysics Data System (ADS)

Marchetti, S.; Sbrana, F.; Toscano, A.; Fratini, E.; Carlà, M.; Vassalli, M.; Tiribilli, B.; Pacini, A.; Gambi, C. M. C.

2011-05-01

The three-dimensional structure and the mechanical properties of a β-connectin fragment from human cardiac muscle, belonging to the I band, from I27 to I34, were investigated by small-angle x-ray scattering (SAXS) and single-molecule force spectroscopy (SMFS). This molecule presents an entropic elasticity behavior, associated to globular domain unfolding, that has been widely studied in the last 10 years. In addition, atomic force microscopy based SMFS experiments suggest that this molecule has an additional elastic regime, for low forces, probably associated to tertiary structure remodeling. From a structural point of view, this behavior is a mark of the fact that the eight domains in the I27-I34 fragment are not independent and they organize in solution, assuming a well-defined three-dimensional structure. This hypothesis has been confirmed by SAXS scattering, both on a diluted and a concentrated sample. Two different models were used to fit the SAXS curves: one assuming a globular shape and one corresponding to an elongated conformation, both coupled with a Coulomb repulsion potential to take into account the protein-protein interaction. Due to the predominance of the structure factor, the effective shape of the protein in solution could not be clearly disclosed. By performing SMFS by atomic force microscopy, mechanical unfolding properties were investigated. Typical sawtooth profiles were obtained and the rupture force of each unfolding domain was estimated. By fitting a wormlike chain model to each peak of the sawtooth profile, the entropic elasticity of octamer was described.

Sop-GPU: accelerating biomolecular simulations in the centisecond timescale using graphics processors.

PubMed

Zhmurov, A; Dima, R I; Kholodov, Y; Barsegov, V

2010-11-01

Theoretical exploration of fundamental biological processes involving the forced unraveling of multimeric proteins, the sliding motion in protein fibers and the mechanical deformation of biomolecular assemblies under physiological force loads is challenging even for distributed computing systems. Using a C(α)-based coarse-grained self organized polymer (SOP) model, we implemented the Langevin simulations of proteins on graphics processing units (SOP-GPU program). We assessed the computational performance of an end-to-end application of the program, where all the steps of the algorithm are running on a GPU, by profiling the simulation time and memory usage for a number of test systems. The ∼90-fold computational speedup on a GPU, compared with an optimized central processing unit program, enabled us to follow the dynamics in the centisecond timescale, and to obtain the force-extension profiles using experimental pulling speeds (v(f) = 1-10 μm/s) employed in atomic force microscopy and in optical tweezers-based dynamic force spectroscopy. We found that the mechanical molecular response critically depends on the conditions of force application and that the kinetics and pathways for unfolding change drastically even upon a modest 10-fold increase in v(f). This implies that, to resolve accurately the free energy landscape and to relate the results of single-molecule experiments in vitro and in silico, molecular simulations should be carried out under the experimentally relevant force loads. This can be accomplished in reasonable wall-clock time for biomolecules of size as large as 10(5) residues using the SOP-GPU package. © 2010 Wiley-Liss, Inc.

Water's role in the force-induced unfolding of ubiquitin.

PubMed

Li, Jingyuan; Fernandez, Julio M; Berne, B J

2010-11-09

In atomic force spectroscopic studies of the elastomeric protein ubiquitin, the β-strands 1-5 serve as the force clamp. Simulations show how the rupture force in the force-induced unfolding depends on the kinetics of water molecule insertion into positions where they can eventually form hydrogen bonding bridges with the backbone hydrogen bonds in the force-clamp region. The intrusion of water into this region is slowed down by the hydrophobic shielding effect of carbonaceous groups on the surface residues of β-strands 1-5, which thereby regulates water insertion prior to hydrogen bond breakage. The experiments show that the unfolding of the mechanically stressed protein is nonexponential due to static disorder. Our simulations show that different numbers and/or locations of bridging water molecules give rise to a long-lived distribution of transition states and static disorder. We find that slowing down the translational (not rotational) motions of the water molecules by increasing the mass of their oxygen atoms, which leaves the force field and thereby the equilibrium structure of the solvent unchanged, increases the average rupture force; however, the early stages of the force versus time behavior are very similar for our "normal" and fictitious "heavy" water models. Finally, we construct six mutant systems to regulate the hydrophobic shielding effect of the surface residues in the force-clamp region. The mutations in the two termini of β-sheets 1-5 are found to determine a preference for different unfolding pathways and change mutant's average rupture force.

Osmotic Pressure Simulations of Amino Acids and Peptides Highlight Potential Routes to Protein Force Field Parameterization

PubMed Central

Miller, Mark S.; Lay, Wesley K.

2016-01-01

Recent molecular dynamics (MD) simulations of proteins have suggested that common force fields overestimate the strength of amino acid interactions in aqueous solution. In an attempt to determine the causes of these effects, we have measured the osmotic coefficients of a number of amino acids using the AMBER ff99SB-ILDN force field with two popular water models, and compared the results with available experimental data. With TIP4P-Ew water, interactions between aliphatic residues agree well with experiment, but interactions of the polar residues serine and threonine are found to be excessively attractive. For all tested amino acids, the osmotic coefficients are lower when the TIP3P water model is used. Additional simulations performed on charged amino acids indicate that the osmotic coefficients are strongly dependent on the parameters assigned to the salt ions, with a reparameterization of the sodium:carboxylate interaction reported by the Aksimentiev group significantly improving description of the osmotic coefficient for glutamate. For five neutral amino acids, we also demonstrate a decrease in solute-solute attractions using the recently reported TIP4P-D water model and using the KBFF force field. Finally, we show that for four two-residue peptides improved agreement with experiment can be achieved by re-deriving the partial charges for each peptide. PMID:27052117

The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces

PubMed Central

Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D; Johnson, Christopher M; Fersht, Alan R

2010-01-01

Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution. PMID:20662005

History, rare, and multiple events of mechanical unfolding of repeat proteins

NASA Astrophysics Data System (ADS)

Sumbul, Fidan; Marchesi, Arin; Rico, Felix

2018-03-01

Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible, and provides larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.

Ingestion of soy-whey blended protein augments sports performance and ameliorates exercise-induced fatigue in a rat exercise model.

PubMed

Ren, Guangxu; Yi, Suqing; Zhang, Hongru; Wang, Jing

2017-02-22

This study sought to determine the effects of soy-whey blended protein supplementation on sports performance and related biochemical parameters after long-term training. After a week of adaptation, eighteen 6-week-old male Wistar rats were randomly assigned to 3 groups: the standard chow diet plus whey protein (Whey) group, the standard chow diet plus soy-whey blended protein (BP) group and the standard chow diet only (control) group. Each group included 6 rats for the seven-week experiment. Before the experiment, the baseline values of body weight, grasping force and time to exhaustion due to the loaded-swimming test were recorded for each group. During the experimental period, all rats performed the loaded-swimming test until exhaustion five days each week. The results showed that the mean maximum grasping force of the BP group significantly increased between the 5 th and the 7 th week (p < 0.05) compared with the other groups. The ingestion of blended protein for 7 weeks significantly increased the mean time to exhaustion due to swimming by 1.5-fold and 1.2-fold compared with the control and Whey groups, respectively. The plasma levels of leucine, isoleucine and valine were significantly higher at 60 min after the blended protein intervention compared with the Whey and control interventions (p < 0.05). Furthermore, the ingestion of soy-whey blended protein enhanced the activities of lactate dehydrogenase and superoxide dismutase and decreased the levels of malondialdehyde in serum. These results collectively suggest that soy-whey blended protein ingestion with resistance exercise can improve sports performance and ameliorate exercise-induced fatigue in rats.

Performance of protein-structure predictions with the physics-based UNRES force field in CASP11.

PubMed

Krupa, Paweł; Mozolewska, Magdalena A; Wiśniewska, Marta; Yin, Yanping; He, Yi; Sieradzan, Adam K; Ganzynkowicz, Robert; Lipska, Agnieszka G; Karczyńska, Agnieszka; Ślusarz, Magdalena; Ślusarz, Rafał; Giełdoń, Artur; Czaplewski, Cezary; Jagieła, Dawid; Zaborowski, Bartłomiej; Scheraga, Harold A; Liwo, Adam

2016-11-01

Participating as the Cornell-Gdansk group, we have used our physics-based coarse-grained UNited RESidue (UNRES) force field to predict protein structure in the 11th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP11). Our methodology involved extensive multiplexed replica exchange simulations of the target proteins with a recently improved UNRES force field to provide better reproductions of the local structures of polypeptide chains. All simulations were started from fully extended polypeptide chains, and no external information was included in the simulation process except for weak restraints on secondary structure to enable us to finish each prediction within the allowed 3-week time window. Because of simplified UNRES representation of polypeptide chains, use of enhanced sampling methods, code optimization and parallelization and sufficient computational resources, we were able to treat, for the first time, all 55 human prediction targets with sizes from 44 to 595 amino acid residues, the average size being 251 residues. Complete structures of six single-domain proteins were predicted accurately, with the highest accuracy being attained for the T0769, for which the CαRMSD was 3.8 Å for 97 residues of the experimental structure. Correct structures were also predicted for 13 domains of multi-domain proteins with accuracy comparable to that of the best template-based modeling methods. With further improvements of the UNRES force field that are now underway, our physics-based coarse-grained approach to protein-structure prediction will eventually reach global prediction capacity and, consequently, reliability in simulating protein structure and dynamics that are important in biochemical processes. Freely available on the web at http://www.unres.pl/ CONTACT: has5@cornell.edu. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Solitons and protein folding: An In Silico experiment

NASA Astrophysics Data System (ADS)

Ilieva, N.; Dai, J.; Sieradzan, A.; Niemi, A.

2015-10-01

Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen's dogma states that the native 3D shape of a protein is completely determined by protein's amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolved problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix-loop-helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics.

Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix.

PubMed

Wijeratne, Sithara S; Martinez, Jerahme R; Grindel, Brian J; Frey, Eric W; Li, Jingqiang; Wang, Liyun; Farach-Carson, Mary C; Kiang, Ching-Hwa

2016-03-01

Perlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone's response to a mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170±20 nm and a diameter of 2-4 nm. Force pulling revealed a strong protein core that can withstand over 100 pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890 pN and a corresponding Young's modulus of 71 MPa. We conclude that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS. Copyright © 2015 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

'Single molecule': theory and experiments, an introduction

PubMed Central

2013-01-01

At scales below micrometers, Brownian motion dictates most of the behaviors. The simple observation of a colloid is striking: a permanent and random motion is seen, whereas inertial forces play a negligible role. This Physics, where velocity is proportional to force, has opened new horizons in biology. The random feature is challenged in living systems where some proteins - molecular motors - have a directed motion whereas their passive behaviors of colloid should lead to a Brownian motion. Individual proteins, polymers of living matter such as DNA, RNA, actin or microtubules, molecular motors, all these objects can be viewed as chains of colloids. They are submitted to shocks from molecules of the solvent. Shapes taken by these biopolymers or dynamics imposed by motors can be measured and modeled from single molecules to their collective effects. Thanks to the development of experimental methods such as optical tweezers, Atomic Force Microscope (AFM), micropipettes, and quantitative fluorescence (such as Förster Resonance Energy Transfer, FRET), it is possible to manipulate these individual biomolecules in an unprecedented manner: experiments allow to probe the validity of models; and a new Physics has thereby emerged with original biological insights. Theories based on statistical mechanics are needed to explain behaviors of these systems. When force-extension curves of these molecules are extracted, the curves need to be fitted with models that predict the deformation of free objects or submitted to a force. When velocity of motors is altered, a quantitative analysis is required to explain the motions of individual molecules under external forces. This lecture will give some elements of introduction to the lectures of the session 'Nanophysics for Molecular Biology'. PMID:24565227

Cytotoxic T cells use mechanical force to potentiate target cell killing

PubMed Central

Basu, Roshni; Whitlock, Benjamin M.; Husson, Julien; Le Floc’h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C.; Huse, Morgan

2016-01-01

SUMMARY The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. PMID:26924577

Further optimization of a hybrid united-atom and coarse-grained force field for folding simulations: Improved backbone hydration and interactions between charged side chains

PubMed Central

Han, Wei; Schulten, Klaus

2012-01-01

PACE, a hybrid force field which couples united-atom protein models with coarse-grained (CG) solvent, has been further optimized, aiming to improve itse ciency for folding simulations. Backbone hydration parameters have been re-optimized based on hydration free energies of polyalanyl peptides through atomistic simulations. Also, atomistic partial charges from all-atom force fields were combined with PACE in order to provide a more realistic description of interactions between charged groups. Using replica exchange molecular dynamics (REMD), ab initio folding using the new PACE has been achieved for seven small proteins (16 – 23 residues) with different structural motifs. Experimental data about folded states, such as their stability at room temperature, melting point and NMR NOE constraints, were also well reproduced. Moreover, a systematic comparison of folding kinetics at room temperature has been made with experiments, through standard MD simulations, showing that the new PACE may speed up the actual folding kinetics 5-10 times. Together with the computational speedup benefited from coarse-graining, the force field provides opportunities to study folding mechanisms. In particular, we used the new PACE to fold a 73-residue protein, 3D, in multiple 10 – 30 μs simulations, to its native states (Cα RMSD ~ 0.34 nm). Our results suggest the potential applicability of the new PACE for the study of folding and dynamics of proteins. PMID:23204949

Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy

NASA Astrophysics Data System (ADS)

Kim, Duckhoe; Sahin, Ozgur

2015-03-01

Scanning probe microscopes can be used to image and chemically characterize surfaces down to the atomic scale. However, the localized tip-sample interactions in scanning probe microscopes limit high-resolution images to the topmost atomic layer of surfaces, and characterizing the inner structures of materials and biomolecules is a challenge for such instruments. Here, we show that an atomic force microscope can be used to image and three-dimensionally reconstruct chemical groups inside a protein complex. We use short single-stranded DNAs as imaging labels that are linked to target regions inside a protein complex, and T-shaped atomic force microscope cantilevers functionalized with complementary probe DNAs allow the labels to be located with sequence specificity and subnanometre resolution. After measuring pairwise distances between labels, we reconstruct the three-dimensional structure formed by the target chemical groups within the protein complex using simple geometric calculations. Experiments with the biotin-streptavidin complex show that the predicted three-dimensional loci of the carboxylic acid groups of biotins are within 2 Å of their respective loci in the corresponding crystal structure, suggesting that scanning probe microscopes could complement existing structural biological techniques in solving structures that are difficult to study due to their size and complexity.

TALEs from a spring--superelasticity of Tal effector protein structures.

PubMed

Flechsig, Holger

2014-01-01

Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA.

TALEs from a Spring – Superelasticity of Tal Effector Protein Structures

PubMed Central

Flechsig, Holger

2014-01-01

Transcription activator-like effectors (TALEs) are DNA-related proteins that recognise and bind specific target sequences to manipulate gene expression. Recently determined crystal structures show that their common architecture reveals a superhelical overall structure that may undergo drastic conformational changes. To establish a link between structure and dynamics in TALE proteins we have employed coarse-grained elastic-network modelling of currently available structural data and implemented a force-probe setup that allowed us to investigate their mechanical behaviour in computer experiments. Based on the measured force-extension curves we conclude that TALEs exhibit superelastic dynamical properties allowing for large-scale global conformational changes along their helical axis, which represents the soft direction in such proteins. For moderate external forcing the TALE models behave like linear springs, obeying Hooke's law, and the investigated structures can be characterised and compared by a corresponding spring constant. We show that conformational flexibility underlying the large-scale motions is not homogeneously distributed over the TALE structure, but instead soft spot residues around which strain is accumulated and which turn out to represent key agents in the transmission of conformational motions are identified. They correspond to the RVD loop residues that have been experimentally determined to play an eminent role in the binding process of target DNA. PMID:25313859

Multidomain proteins under force

NASA Astrophysics Data System (ADS)

Valle-Orero, Jessica; Andrés Rivas-Pardo, Jaime; Popa, Ionel

2017-04-01

Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91—two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins—ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

Aligned Immobilization of Proteins Using AC Electric Fields.

PubMed

Laux, Eva-Maria; Knigge, Xenia; Bier, Frank F; Wenger, Christian; Hölzel, Ralph

2016-03-01

Protein molecules are aligned and immobilized from solution by AC electric fields. In a single-step experiment, the enhanced green fluorescent proteins are immobilized on the surface as well as at the edges of planar nanoelectrodes. Alignment is found to follow the molecules' geometrical shape with their longitudinal axes parallel to the electric field. Simultaneous dielectrophoretic attraction and AC electroosmotic flow are identified as the dominant forces causing protein movement and alignment. Molecular orientation is determined by fluorescence microscopy based on polarized excitation of the proteins' chromophores. The chromophores' orientation with respect to the whole molecule supports X-ray crystal data. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Revealing Abrupt and Spontaneous Ruptures of Protein Native Structure under picoNewton Compressive Force Manipulation.

PubMed

Chowdhury, S Roy; Cao, Jin; He, Yufan; Lu, H Peter

2018-03-27

Manipulating protein conformations for exploring protein structure-function relationship has shown great promise. Although protein conformational changes under pulling force manipulation have been extensively studied, protein conformation changes under a compressive force have not been explored quantitatively. The latter is even more biologically significant and relevant in revealing protein functions in living cells associated with protein crowdedness, distribution fluctuations, and cell osmotic stress. Here we report our experimental observations on abrupt ruptures of protein native structures under compressive force, demonstrated and studied by single-molecule AFM-FRET spectroscopic nanoscopy. Our results show that the protein ruptures are abrupt and spontaneous events occurred when the compressive force reaches a threshold of 12-75 pN, a force amplitude accessible from thermal fluctuations in a living cell. The abrupt ruptures are sensitive to local environment, likely a general and important pathway of protein unfolding in living cells.

Consistent free energy landscapes and thermodynamic properties of small proteins based on a single all-atom force field employing an implicit solvation.

PubMed

Kim, Eunae; Jang, Soonmin; Pak, Youngshang

2007-10-14

We have attempted to improve the PARAM99 force field in conjunction with the generalized Born (GB) solvation model with a surface area correction for more consistent protein folding simulations. For this purpose, using an extended alphabeta training set of five well-studied molecules with various folds (alpha, beta, and betabetaalpha), a previously modified version of PARAM99/GBSA is further refined, such that all native states of the five training species correspond to their lowest free energy minimum states. The resulting modified force field (PARAM99MOD5/GBSA) clearly produces reasonably acceptable conformational free energy surfaces of the training set with correct identifications of their native states in the free energy minimum states. Moreover, due to its well-balanced nature, this new force field is expected to describe secondary structure propensities of diverse folds in a more consistent manner. Remarkably, temperature dependent behaviors simulated with the current force field are in good agreement with the experiment. This agreement is a significant improvement over the existing standard all-atom force fields. In addition, fundamentally important thermodynamic quantities, such as folding enthalpy (DeltaH) and entropy (DeltaS), agree reasonably well with the experimental data.

Towards force spectroscopy of single tip-link bonds

NASA Astrophysics Data System (ADS)

Koussa, Mounir A.; Sotomayor, Marcos; Wong, Wesley P.; Corey, David P.

2015-12-01

Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. Molecular dynamics simulations predict that the tip-link bond is mechanically strong enough to withstand forces in hair cells, but its experimentally determined strength is unknown. We have developed molecular tools to facilitate single-molecule force spectroscopy on the tip link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip link nanoswitches are designed to provide a signature force-extension profile. This molecular signature should allow us to identify single-molecule rupture events in pulling experiments.

DelPhiForce web server: electrostatic forces and energy calculations and visualization.

PubMed

Li, Lin; Jia, Zhe; Peng, Yunhui; Chakravorty, Arghya; Sun, Lexuan; Alexov, Emil

2017-11-15

Electrostatic force is an essential component of the total force acting between atoms and macromolecules. Therefore, accurate calculations of electrostatic forces are crucial for revealing the mechanisms of many biological processes. We developed a DelPhiForce web server to calculate and visualize the electrostatic forces at molecular level. DelPhiForce web server enables modeling of electrostatic forces on individual atoms, residues, domains and molecules, and generates an output that can be visualized by VMD software. Here we demonstrate the usage of the server for various biological problems including protein-cofactor, domain-domain, protein-protein, protein-DNA and protein-RNA interactions. The DelPhiForce web server is available at: http://compbio.clemson.edu/delphi-force. delphi@clemson.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Probing the nanoscale interaction forces and elastic properties of organic and inorganic materials using force-distance (F-D) spectroscopy

NASA Astrophysics Data System (ADS)

Vincent, Abhilash

Due to their therapeutic applications such as radical scavenging, MRI contrast imaging, Photoluminescence imaging, drug delivery, etc., nanoparticles (NPs) have a significant importance in bio-nanotechnology. The reason that prevents the utilizing NPs for drug delivery in medical field is mostly due to their biocompatibility issues (incompatibility can lead to toxicity and cell death). Changes in the surface conditions of NPs often lead to NP cytotoxicity. Investigating the role of NP surface properties (surface charges and surface chemistry) on their interactions with biomolecules (Cells, protein and DNA) could enhance the current understanding of NP cytotoxicity. Hence, it is highly beneficial to the nanotechnology community to bring more attention towards the enhancement of surface properties of NPs to make them more biocompatible and less toxic to biological systems. Surface functionalization of NPs using specific ligand biomolecules have shown to enhance the protein adsorption and cellular uptake through more favorable interaction pathways. Cerium oxide NPs (CNPs also known as nanoceria) are potential antioxidants in cell culture models and understanding the nature of interaction between cerium oxide NPs and biological proteins and cells are important due to their therapeutic application (especially in site specific drug delivery systems). The surface charges and surface chemistry of CNPs play a major role in protein adsorption and cellular uptake. Hence, by tuning the surface charges and by selecting proper functional molecules on the surface, CNPs exhibiting strong adhesion to biological materials can be prepared. By probing the nanoscale interaction forces acting between CNPs and protein molecules using Atomic Force Microscopy (AFM) based force-distance (F-D) spectroscopy, the mechanism of CNP-protein adsorption and CNP cellular uptake can be understood more quantitatively. The work presented in this dissertation is based on the application of AFM in studying the interaction forces as well as the mechanical properties of nanobiomaterials. The research protocol employed in the earlier part of the dissertation is specifically aimed to understand the operation of F-D spectroscopy technique. The elastic properties of thin films of silicon dioxide NPs were investigated using F-D spectroscopy in the high force regime of few 100 nN to 1 microN. Here, sol-gel derived porous nanosilica thin films of varying surface morphology, particle size and porosity were prepared through acid and base catalyzed process. AFM nanoindentation experiments were conducted on these films using the F-D spectroscopy mode and the nanoscale elastic properties of these films were evaluated. The major contribution of this dissertation is a study exploring the interaction forces acting between CNPs and transferrin proteins in picoNewton scale regime using the force-distance spectroscopy technique. This study projects the importance of obtaining appropriate surface charges and surface chemistry so that the NP can exhibit enhanced protein adsorption and NP cellular uptake.

Force spectroscopy of biomolecular folding and binding: theory meets experiment

NASA Astrophysics Data System (ADS)

Dudko, Olga

2015-03-01

Conformational transitions in biological macromolecules usually serve as the mechanism that brings biomolecules into their working shape and enables their biological function. Single-molecule force spectroscopy probes conformational transitions by applying force to individual macromolecules and recording their response, or ``mechanical fingerprints,'' in the form of force-extension curves. However, how can we decode these fingerprints so that they reveal the kinetic barriers and the associated timescales of a biological process? I will present an analytical theory of the mechanical fingerprints of macromolecules. The theory is suitable for decoding such fingerprints to extract the barriers and timescales. The application of the theory will be illustrated through recent studies on protein-DNA interactions and the receptor-ligand complexes involved in blood clot formation.

The Biotin/Avidin complex adhesion force

NASA Astrophysics Data System (ADS)

Balsera, Manel A.; Izrailev, Sergei; Stepaniants, Sergey; Oono, Yoshitsugu; Schulten, Klaus

1997-03-01

The vitamin Biotin and the protein avidin form one of the strongest non-covalent bonds between biological molecules. We have performed molecular and stochastic dynamic modeling of the unbinding of this complex(Izrailev et al., Biophysical Journal, In press). These simulations provide insight into the effect of particular residues and water on the tight binding of the system. With the aid of simple phenomenological models we have related qualitatively our results to Atomic Force Microscopy adhesion force measurements (E.-L. Florin, V. T. Moy and H. E. Gaub Science) 264:415-417 and kinetic dissociation experiments( A. Chilcotti and P. S. Stayton, J. Am. Chem. Soc.) 117:10622-10628. We will discuss the difficulties preventing a more quantitative understanding of the unbinding force and kinetics.

Optimizing the Immuno-Surface Characteristics for Bio-Sensors and Filters Through Modeling and Experiments

DTIC Science & Technology

2005-06-01

immobilization of antibodies o Adsorbed, aminophase, heterobifunctional crosslinkers (GMBS, BMPS, EMCS) o GMBS attaches the most antibodies o ProteinA ...play a role in getting the antigen close enough to the immuno-surface to potentially interact as well as the short range molecular forces that

Forces from the Portal Govern the Late-Stage DNA Transport in a Viral DNA Packaging Nanomotor.

PubMed

Jing, Peng; Burris, Benjamin; Zhang, Rong

2016-07-12

In the Phi29 bacteriophage, the DNA packaging nanomotor packs its double-stranded DNA genome into the virus capsid. At the late stage of DNA packaging, the negatively charged genome is increasingly compacted at a higher density in the capsid with a higher internal pressure. During the process, two Donnan effects, osmotic pressure and Donnan equilibrium potentials, are significantly amplified, which, in turn, affect the channel activity of the portal protein, GP10, embedded in the semipermeable capsid shell. In the research, planar lipid bilayer experiments were used to study the channel activities of the viral protein. The Donnan effect on the conformational changes of the viral protein was discovered, indicating GP10 may not be a static channel at the late stage of DNA packaging. Due to the conformational changes, GP10 may generate electrostatic forces that govern the DNA transport. For the section of the genome DNA that remains outside of the connector channel, a strong repulsive force from the viral protein would be generated against the DNA entry; however, for the section of the genome DNA within the channel, the portal protein would become a Brownian motor, which adopts the flash Brownian ratchet mechanism to pump the DNA against the increasingly built-up internal pressure (up to 20 atm) in the capsid. Therefore, the DNA transport in the nanoscale viral channel at the late stage of DNA packaging could be a consequence of Brownian movement of the genomic DNA, which would be rectified and harnessed by the forces from the interior wall of the viral channel under the influence of the Donnan effect. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Detailed analysis of grid-based molecular docking: A case study of CDOCKER-A CHARMm-based MD docking algorithm.

PubMed

Wu, Guosheng; Robertson, Daniel H; Brooks, Charles L; Vieth, Michal

2003-10-01

The influence of various factors on the accuracy of protein-ligand docking is examined. The factors investigated include the role of a grid representation of protein-ligand interactions, the initial ligand conformation and orientation, the sampling rate of the energy hyper-surface, and the final minimization. A representative docking method is used to study these factors, namely, CDOCKER, a molecular dynamics (MD) simulated-annealing-based algorithm. A major emphasis in these studies is to compare the relative performance and accuracy of various grid-based approximations to explicit all-atom force field calculations. In these docking studies, the protein is kept rigid while the ligands are treated as fully flexible and a final minimization step is used to refine the docked poses. A docking success rate of 74% is observed when an explicit all-atom representation of the protein (full force field) is used, while a lower accuracy of 66-76% is observed for grid-based methods. All docking experiments considered a 41-member protein-ligand validation set. A significant improvement in accuracy (76 vs. 66%) for the grid-based docking is achieved if the explicit all-atom force field is used in a final minimization step to refine the docking poses. Statistical analysis shows that even lower-accuracy grid-based energy representations can be effectively used when followed with full force field minimization. The results of these grid-based protocols are statistically indistinguishable from the detailed atomic dockings and provide up to a sixfold reduction in computation time. For the test case examined here, improving the docking accuracy did not necessarily enhance the ability to estimate binding affinities using the docked structures. Copyright 2003 Wiley Periodicals, Inc.

Systems Mechanobiology: Tension-Inhibited Protein Turnover Is Sufficient to Physically Control Gene Circuits

PubMed Central

Dingal, P.C. Dave P.; Discher, Dennis E.

2014-01-01

Mechanotransduction pathways convert forces that stress and strain structures within cells into gene expression levels that impact development, homeostasis, and disease. The levels of some key structural proteins in the nucleus, cytoskeleton, or extracellular matrix have been recently reported to scale with tissue- and cell-level forces or mechanical properties such as stiffness, and so the mathematics of mechanotransduction becomes important to understand. Here, we show that if a given structural protein positively regulates its own gene expression, then stresses need only inhibit degradation of that protein to achieve stable, mechanosensitive gene expression. This basic use-it-or-lose-it module is illustrated by application to meshworks of nuclear lamin A, minifilaments of myosin II, and extracellular matrix collagen fibers—all of which possess filamentous coiled-coil/supercoiled structures. Past experiments not only suggest that tension suppresses protein degradation mediated and/or initiated by various enzymes but also that transcript levels vary with protein levels because key transcription factors are regulated by these structural proteins. Coupling between modules occurs within single cells and between cells in tissue, as illustrated during embryonic heart development where cardiac fibroblasts make collagen that cardiomyocytes contract. With few additional assumptions, the basic module has sufficient physics to control key structural genes in both development and disease. PMID:25468352

Correlation between surface morphology and surface forces of protein A adsorbed on mica.

PubMed Central

Ohnishi, S; Murata, M; Hato, M

1998-01-01

We have investigated the morphology and surface forces of protein A adsorbed on mica surface in the protein solutions of various concentrations. The force-distance curves, measured with a surface force apparatus (SFA), were interpreted in terms of two different regimens: a "large-distance" regimen in which an electrostatic double-layer force dominates, and an "adsorbed layer" regimen in which a force of steric origin dominates. To further clarify the forces of steric origin, the surface morphology of the adsorbed protein layer was investigated with an atomic force microscope (AFM) because the steric repulsive forces are strongly affected by the adsorption mode of protein A molecules on mica. At lower protein concentrations (2 ppm, 10 ppm), protein A molecules were adsorbed "side-on" parallel to the mica surfaces, forming a monolayer of approximately 2.5 nm. AFM images at higher concentrations (30 ppm, 100 ppm) showed protruding structures over the monolayer, which revealed that the adsorbed protein A molecules had one end oriented into the solution, with the remainder of each molecule adsorbed side-on to the mica surface. These extending ends of protein A overlapped each other and formed a "quasi-double layer" over the mica surface. These AFM images proved the existence of a monolayer of protein A molecules at low concentrations and a "quasi-double layer" with occasional protrusions at high concentrations, which were consistent with the adsorption mode observed in the force-distance curves. PMID:9449346

iATTRACT: simultaneous global and local interface optimization for protein-protein docking refinement.

PubMed

Schindler, Christina E M; de Vries, Sjoerd J; Zacharias, Martin

2015-02-01

Protein-protein interactions are abundant in the cell but to date structural data for a large number of complexes is lacking. Computational docking methods can complement experiments by providing structural models of complexes based on structures of the individual partners. A major caveat for docking success is accounting for protein flexibility. Especially, interface residues undergo significant conformational changes upon binding. This limits the performance of docking methods that keep partner structures rigid or allow limited flexibility. A new docking refinement approach, iATTRACT, has been developed which combines simultaneous full interface flexibility and rigid body optimizations during docking energy minimization. It employs an atomistic molecular mechanics force field for intermolecular interface interactions and a structure-based force field for intramolecular contributions. The approach was systematically evaluated on a large protein-protein docking benchmark, starting from an enriched decoy set of rigidly docked protein-protein complexes deviating by up to 15 Å from the native structure at the interface. Large improvements in sampling and slight but significant improvements in scoring/discrimination of near native docking solutions were observed. Complexes with initial deviations at the interface of up to 5.5 Å were refined to significantly better agreement with the native structure. Improvements in the fraction of native contacts were especially favorable, yielding increases of up to 70%. © 2014 Wiley Periodicals, Inc.

Discrete persistent-chain model for protein binding on DNA.

PubMed

Lam, Pui-Man; Zhen, Yi

2011-04-01

We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society

An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules

PubMed Central

Nicholas, Matthew P.; Rao, Lu; Gennerich, Arne

2014-01-01

Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments. PMID:24633799

In Silico Measurements of Twist and Bend Moduli for β-Solenoid Protein Self-Assembly Units.

PubMed

Heinz, Leonard P; Ravikumar, Krishnakumar M; Cox, Daniel L

2015-05-13

We compute potentials of mean force for bend and twist deformations via force pulling and umbrella sampling experiments for four β-solenoid proteins (BSPs) that show promise in nanotechnology applications. In all cases, we find quasi-Hooke's law behavior until the point of rupture. Bending moduli show modest anisotropy for two-sided and three-sided BSPs, and little anisotropy for a four-sided BSP. There is a slight clockwise/counterclockwise asymmetry in the twist potential of mean force, showing greater stiffness when the applied twist follows the intrinsic twist. When we extrapolate to beam theory appropriate for amyloid fibrils of the BSPs, we find bend/twist moduli which are somewhat smaller than those in the literature for other amyloid fibrils. Twist persistence lengths are on the order of a micron, and bend persistence lengths are several microns. Provided the intrinsic twist can be reversed, these results support the usage of BSPs in biomaterials applications.

Dynamic information for cardiotoxin protein desorption from a methyl-terminated self-assembled monolayer using steered molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Hung, Shih-Wei; Hsiao, Pai-Yi; Chieng, Ching-Chang

2011-05-01

Dynamic information, such as force, structural change, interaction energy, and potential of mean force (PMF), about the desorption of a single cardiotoxin (CTX) protein from a methyl-terminated self-assembled monolayer (SAM) surface was investigated by means of steered molecular dynamics (SMD) simulations. The simulation results indicated that Loop I is the first loop to depart from the SAM surface, which is in good agreement with the results of the nuclear magnetic resonance spectroscopy experiment. The free energy landscape and the thermodynamic force of the CTX desorption process was represented by the PMF and by the derivative of PMF with respect to distance, respectively. By applying Jarzynski's equality, the PMF can be reconstructed from the SMD simulation. The PMFs, calculated by different estimators based upon Jarzynski's equality, were compared with the conventional umbrella sampling method. The best estimation was obtained by using the fluctuation-dissipation estimator with a pulling velocity of v = 0.25 nm/ns for the present study.

Surface force measurements and simulations of mussel-derived peptide adhesives on wet organic surfaces.

PubMed

Levine, Zachary A; Rapp, Michael V; Wei, Wei; Mullen, Ryan Gotchy; Wu, Chun; Zerze, Gül H; Mittal, Jeetain; Waite, J Herbert; Israelachvili, Jacob N; Shea, Joan-Emma

2016-04-19

Translating sticky biological molecules-such as mussel foot proteins (MFPs)-into synthetic, cost-effective underwater adhesives with adjustable nano- and macroscale characteristics requires an intimate understanding of the glue's molecular interactions. To help facilitate the next generation of aqueous adhesives, we performed a combination of surface forces apparatus (SFA) measurements and replica-exchange molecular dynamics (REMD) simulations on a synthetic, easy to prepare, Dopa-containing peptide (MFP-3s peptide), which adheres to organic surfaces just as effectively as its wild-type protein analog. Experiments and simulations both show significant differences in peptide adsorption on CH3-terminated (hydrophobic) and OH-terminated (hydrophilic) self-assembled monolayers (SAMs), where adsorption is strongest on hydrophobic SAMs because of orientationally specific interactions with Dopa. Additional umbrella-sampling simulations yield free-energy profiles that quantitatively agree with SFA measurements and are used to extract the adhesive properties of individual amino acids within the context of MFP-3s peptide adhesion, revealing a delicate balance between van der Waals, hydrophobic, and electrostatic forces.

Mechanics of composite actin networks: in vitro and cellular perspectives

NASA Astrophysics Data System (ADS)

Upadhyaya, Arpita

2014-03-01

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

Characterization of fiber-forming peptides and proteins by means of atomic force microscopy.

PubMed

Creasey, Rhiannon G; Gibson, Christopher T; Voelcker, Nicolas H

2012-05-01

The atomic force microscope (AFM) is widely used in biological sciences due to its ability to perform imaging experiments at high resolution in a physiological environment, without special sample preparation such as fixation or staining. AFM is unique, in that it allows single molecule information of mechanical properties and molecular recognition to be gathered. This review sets out to identify methodological applications of AFM for characterization of fiber-forming proteins and peptides. The basics of AFM operation are detailed, with in-depth information for any life scientist to get a grasp on AFM capabilities. It also briefly describes antibody recognition imaging and mapping of nanomechanical properties on biological samples. Subsequently, examples of AFM application to fiber-forming natural proteins, and fiber-forming synthetic peptides are given. Here, AFM is used primarily for structural characterization of fibers in combination with other techniques, such as circular dichroism and fluorescence spectroscopy. More recent developments in antibody recognition imaging to identify constituents of protein fibers formed in human disease are explored. This review, as a whole, seeks to encourage the life scientists dealing with protein aggregation phenomena to consider AFM as a part of their research toolkit, by highlighting the manifold capabilities of this technique.

Controlled immobilization of His-tagged proteins for protein-ligand interaction experiments using Ni²⁺-NTA layer on glass surfaces.

PubMed

Cherkouk, Charaf; Rebohle, Lars; Lenk, Jens; Keller, Adrian; Ou, Xin; Laube, Markus; Neuber, Christin; Haase-Kohn, Cathleen; Skorupa, Wolfgang; Pietzsch, Jens

2015-01-01

Gold surfaces functionalized with nickel-nitrilotriacetic acid (Ni²⁺-NTA) as self-assembled monolayers (SAM) to immobilize histidine (His)-tagged biomolecules are broadly reported in the literature. However, the increasing demand of using microfluidic systems and biosensors takes more and more advantage on silicon technology which provides dedicated glass surfaces and substantially allows cost and resource savings. Here we present a novel method for the controlled oriented immobilization of His-tagged proteins on glass surfaces functionalized with a Ni²⁺-NTA layer. Exemplarily, the protein pattern morphology after immobilization on the Ni²⁺-NTA layer of His6-tagged soluble receptor for advanced glycation endproducts (sRAGE) was investigated and compared to non-oriented immobilization of sRAGE on amino SAM by using scanning electron microscopy (SEM). Moreover, we demonstrated interaction of immobilized sRAGE with three structurally different ligands, S100A12, S100A4, and glycated low density lipoproteins (glycLDL), by means of peak-force tapping atomic force microscopy (PF-AFM). We showed a clear discrimination of different protein-ligand orientations by differential height measurements.

Effect of protein solution components in the adsorption of Herbaspirillum seropedicae GlnB protein on mica.

PubMed

Ferreira, Cecília F G; Benelli, Elaine M; Klein, Jorge J; Schreiner, Wido; Camargo, Paulo C

2009-10-15

The adsorption of proteins and its buffer solution on mica surfaces was investigated by atomic force microscopy (AFM). Different salt concentration of the Herbaspirillum seropedicae GlnB protein (GlnB-Hs) solution deposited on mica was investigated. This protein is a globular, soluble homotrimer (36kDa), member of PII-like proteins family involved in signal transducing in prokaryote. Supramolecular structures were formed when this protein was deposited onto bare mica surface. The topographic AFM images of the GlnB-Hs films showed that at high salt concentration the supramolecular structures are spherical-like, instead of the typical doughnut-like shape for low salt concentration. AFM images of NaCl and Tris from the buffer solution showed structures with the same pattern as those observed for high salt protein solution, misleading the image interpretation. XPS experiments showed that GlnB protein film covers the mica surface without chemical reaction.

Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase.

PubMed

Scholl, Zackary N; Yang, Weitao; Marszalek, Piotr E

2017-05-09

Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarse-grained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Current Status of Protein Force Fields for Molecular Dynamics

PubMed Central

Lopes, Pedro E.M.; Guvench, Olgun

2015-01-01

Summary The current status of classical force fields for proteins is reviewed. These include additive force fields as well as the latest developments in the Drude and AMOEBA polarizable force fields. Parametrization strategies developed specifically for the Drude force field are described and compared with the additive CHARMM36 force field. Results from molecular simulations of proteins and small peptides are summarized to illustrate the performance of the Drude and AMOEBA force fields. PMID:25330958

Adhesion of bubbles and drops to solid surfaces, and anisotropic surface tensions studied by capillary meniscus dynamometry.

PubMed

Danov, Krassimir D; Stanimirova, Rumyana D; Kralchevsky, Peter A; Marinova, Krastanka G; Stoyanov, Simeon D; Blijdenstein, Theodorus B J; Cox, Andrew R; Pelan, Eddie G

2016-07-01

Here, we review the principle and applications of two recently developed methods: the capillary meniscus dynamometry (CMD) for measuring the surface tension of bubbles/drops, and the capillary bridge dynamometry (CBD) for quantifying the bubble/drop adhesion to solid surfaces. Both methods are based on a new data analysis protocol, which allows one to decouple the two components of non-isotropic surface tension. For an axisymmetric non-fluid interface (e.g. bubble or drop covered by a protein adsorption layer with shear elasticity), the CMD determines the two different components of the anisotropic surface tension, σs and σφ, which are acting along the "meridians" and "parallels", and vary throughout the interface. The method uses data for the instantaneous bubble (drop) profile and capillary pressure, but the procedure for data processing is essentially different from that of the conventional drop shape analysis (DSA) method. In the case of bubble or drop pressed against a substrate, which forms a capillary bridge, the CBD method allows one to determine also the capillary-bridge force for both isotropic (fluid) and anisotropic (solidified) adsorption layers. The experiments on bubble (drop) detachment from the substrate show the existence of a maximal pulling force, Fmax, that can be resisted by an adherent fluid particle. Fmax can be used to quantify the strength of adhesion of bubbles and drops to solid surfaces. Its value is determined by a competition of attractive transversal tension and repulsive disjoining pressure forces. The greatest Fmax values have been measured for bubbles adherent to glass substrates in pea-protein solutions. The bubble/wall adhesion is lower in solutions containing the protein HFBII hydrophobin, which could be explained with the effect of sandwiched protein aggregates. The applicability of the CBD method to emulsion systems is illustrated by experiments with soybean-oil drops adherent to hydrophilic and hydrophobic substrates in egg yolk solutions. The results reveal how the interfacial rigidity, as well as the bubble/wall and drop/wall adhesion forces, can be quantified and controlled in relation to optimizing the properties of foams and emulsions. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular Dynamics Simulations of Intrinsically Disordered Proteins: On the Accuracy of the TIP4P-D Water Model and the Representativeness of Protein Disorder Models.

PubMed

Henriques, João; Skepö, Marie

2016-07-12

Here, we first present a follow-up to a previous work by our group on the problematic of molecular dynamics simulations of intrinsically disordered proteins (IDPs) [ Henriques et al. J. Chem. Theory Comput. 2015 , 11 , 3420 - 3431 ], using the recently developed TIP4P-D water model. When used in conjunction with the standard AMBER ff99SB-ILDN force field and applied to the simulation of Histatin 5, our IDP model, we obtain results which are in excellent agreement with the best performing IDP-suitable force field from the earlier study and with experiment. We then assess the representativeness of the IDP models used in these and similar studies, finding that most are too short in comparison to the average IDP and contain a bias toward hydrophilic amino acid residues. Moreover, several key order- and disorder-promoting residues are also found to be misrepresented. It seems appropriate for future studies to address these issues.

Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

PubMed

Basu, Roshni; Whitlock, Benjamin M; Husson, Julien; Le Floc'h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C; Huse, Morgan

2016-03-24

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. Copyright © 2016 Elsevier Inc. All rights reserved.

Global analysis of protein folding using massively parallel design, synthesis and testing

PubMed Central

Rocklin, Gabriel J.; Chidyausiku, Tamuka M.; Goreshnik, Inna; Ford, Alex; Houliston, Scott; Lemak, Alexander; Carter, Lauren; Ravichandran, Rashmi; Mulligan, Vikram K.; Chevalier, Aaron; Arrowsmith, Cheryl H.; Baker, David

2017-01-01

Proteins fold into unique native structures stabilized by thousands of weak interactions that collectively overcome the entropic cost of folding. Though these forces are “encoded” in the thousands of known protein structures, “decoding” them is challenging due to the complexity of natural proteins that have evolved for function, not stability. Here we combine computational protein design, next-generation gene synthesis, and a high-throughput protease susceptibility assay to measure folding and stability for over 15,000 de novo designed miniproteins, 1,000 natural proteins, 10,000 point-mutants, and 30,000 negative control sequences, identifying over 2,500 new stable designed proteins in four basic folds. This scale—three orders of magnitude greater than that of previous studies of design or folding—enabled us to systematically examine how sequence determines folding and stability in uncharted protein space. Iteration between design and experiment increased the design success rate from 6% to 47%, produced stable proteins unlike those found in nature for topologies where design was initially unsuccessful, and revealed subtle contributions to stability as designs became increasingly optimized. Our approach achieves the long-standing goal of a tight feedback cycle between computation and experiment, and promises to transform computational protein design into a data-driven science. PMID:28706065

Precise control of surface electrostatic forces on polymer brush layers with opposite charges for resistance to protein adsorption.

PubMed

Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

2016-10-01

Various molecular interaction forces are generated during protein adsorption process on material surfaces. Thus, it is necessary to control them to suppress protein adsorption and the subsequent cell and tissue responses. A series of binary copolymer brush layers were prepared via surface-initiated atom transfer radical polymerization, by mixing the cationic monomer unit and anionic monomer unit randomly in various ratios. Surface characterization revealed that the constructed copolymer brush layers exhibited an uniform super-hydrophilic nature and different surface potentials. The strength of the electrostatic interaction forces operating on these mixed-charge copolymer brush surfaces was evaluated quantitatively using force-versus-distance (f-d) curve measurements by atomic force microscopy (AFM) and probes modified by negatively charged carboxyl groups or positively charged amino groups. The electrostatic interaction forces were determined based on the charge ratios of the copolymer brush layers. Notably, the surface containing equivalent cationic/anionic monomer units hardly interacted with both the charged groups. Furthermore, the protein adsorption force and the protein adsorption mass on these surfaces were examined by AFM f-d curve measurement and surface plasmon resonance measurement, respectively. To clarify the influence of the electrostatic interaction on the protein adsorption behavior on the surface, three kinds of proteins having negative, positive, and relatively neutral net charges under physiological conditions were used in this study. We quantitatively demonstrated that the amount of adsorbed proteins on the surfaces would have a strong correlation with the strength of surface-protein interaction forces, and that the strength of surface-protein interaction forces would be determined from the combination between the properties of the electrostatic interaction forces on the surfaces and the charge properties of the proteins. Especially, the copolymer brush surface composed of equivalent cationic/anionic monomer units exhibited no significant interaction forces, and dramatically suppressed the adsorption of proteins regardless of their charge properties. We conclude that the established methodology could elucidate relationship between the protein adsorption behavior and molecular interaction, especially the electrostatic interaction forces, and demonstrated that the suppression of the electrostatic interactions with the ionic functional groups would be important for the development of new polymeric biomaterials with a high repellency of protein adsorption. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nanoindentation studies of full and empty viral capsids and the effects of capsid protein mutations on elasticity and strength

NASA Astrophysics Data System (ADS)

Michel, J. P.; Ivanovska, I. L.; Gibbons, M. M.; Klug, W. S.; Knobler, C. M.; Wuite, G. J. L.; Schmidt, C. F.

2006-04-01

The elastic properties of capsids of the cowpea chlorotic mottle virus have been examined at pH 4.8 by nanoindentation measurements with an atomic force microscope. Studies have been carried out on WT capsids, both empty and containing the RNA genome, and on full capsids of a salt-stable mutant and empty capsids of the subE mutant. Full capsids resisted indentation more than empty capsids, but all of the capsids were highly elastic. There was an initial reversible linear regime that persisted up to indentations varying between 20% and 30% of the diameter and applied forces of 0.6-1.0 nN; it was followed by a steep drop in force that is associated with irreversible deformation. A single point mutation in the capsid protein increased the capsid stiffness. The experiments are compared with calculations by finite element analysis of the deformation of a homogeneous elastic thick shell. These calculations capture the features of the reversible indentation region and allow Young's moduli and relative strengths to be estimated for the empty capsids. atomic force microscopy | cowpea chlorotic mottle virus | finite element analysis | biomechanics

Stabilising the Herpes Simplex Virus capsid by DNA packaging

NASA Astrophysics Data System (ADS)

Wuite, Gijs; Radtke, Kerstin; Sodeik, Beate; Roos, Wouter

2009-03-01

Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids.

Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein

NASA Astrophysics Data System (ADS)

Guinn, Emily J.; Jagannathan, Bharat; Marqusee, Susan

2015-04-01

A fundamental question in protein folding is whether proteins fold through one or multiple trajectories. While most experiments indicate a single pathway, simulations suggest proteins can fold through many parallel pathways. Here, we use a combination of chemical denaturant, mechanical force and site-directed mutations to demonstrate the presence of multiple unfolding pathways in a simple, two-state folding protein. We show that these multiple pathways have structurally different transition states, and that seemingly small changes in protein sequence and environment can strongly modulate the flux between the pathways. These results suggest that in vivo, the crowded cellular environment could strongly influence the mechanisms of protein folding and unfolding. Our study resolves the apparent dichotomy between experimental and theoretical studies, and highlights the advantage of using a multipronged approach to reveal the complexities of a protein's free-energy landscape.

i3Drefine software for protein 3D structure refinement and its assessment in CASP10.

PubMed

Bhattacharya, Debswapna; Cheng, Jianlin

2013-01-01

Protein structure refinement refers to the process of improving the qualities of protein structures during structure modeling processes to bring them closer to their native states. Structure refinement has been drawing increasing attention in the community-wide Critical Assessment of techniques for Protein Structure prediction (CASP) experiments since its addition in 8(th) CASP experiment. During the 9(th) and recently concluded 10(th) CASP experiments, a consistent growth in number of refinement targets and participating groups has been witnessed. Yet, protein structure refinement still remains a largely unsolved problem with majority of participating groups in CASP refinement category failed to consistently improve the quality of structures issued for refinement. In order to alleviate this need, we developed a completely automated and computationally efficient protein 3D structure refinement method, i3Drefine, based on an iterative and highly convergent energy minimization algorithm with a powerful all-atom composite physics and knowledge-based force fields and hydrogen bonding (HB) network optimization technique. In the recent community-wide blind experiment, CASP10, i3Drefine (as 'MULTICOM-CONSTRUCT') was ranked as the best method in the server section as per the official assessment of CASP10 experiment. Here we provide the community with free access to i3Drefine software and systematically analyse the performance of i3Drefine in strict blind mode on the refinement targets issued in CASP10 refinement category and compare with other state-of-the-art refinement methods participating in CASP10. Our analysis demonstrates that i3Drefine is only fully-automated server participating in CASP10 exhibiting consistent improvement over the initial structures in both global and local structural quality metrics. Executable version of i3Drefine is freely available at http://protein.rnet.missouri.edu/i3drefine/.

Metrics that differentiate the origins of osmolyte effects on protein stability: a test of the surface tension proposal.

PubMed

Auton, Matthew; Ferreon, Allan Chris M; Bolen, D Wayne

2006-09-01

Osmolytes that are naturally selected to protect organisms against environmental stresses are known to confer stability to proteins via preferential exclusion from protein surfaces. Solvophobicity, surface tension, excluded volume, water structure changes and electrostatic repulsion are all examples of forces proposed to account for preferential exclusion and the ramifications exclusion has on protein properties. What has been lacking is a systematic way of determining which force(s) is(are) responsible for osmolyte effects. Here, we propose the use of two experimental metrics for assessing the abilities of various proposed forces to account for osmolyte-mediated effects on protein properties. Metric 1 requires prediction of the experimentally determined ability of the osmolyte to bring about folding/unfolding resulting from the application of the force in question (i.e. prediction of the m-value of the protein in osmolyte). Metric 2 requires prediction of the experimentally determined ability of the osmolyte to contract or expand the Stokes radius of the denatured state resulting from the application of the force. These metrics are applied to test separate claims that solvophobicity/solvophilicity and surface tension are driving forces for osmolyte-induced effects on protein stability. The results show clearly that solvophobic/solvophilic forces readily account for protein stability and denatured state dimensional effects, while surface tension alone fails to do so. The agreement between experimental and predicted m-values involves both positive and negative m-values for three different proteins, and as many as six different osmolytes, illustrating that the tests are robust and discriminating. The ability of the two metrics to distinguish which forces account for the effects of osmolytes on protein properties and which do not, provides a powerful means of investigating the origins of osmolyte-protein effects.

How accurately do force fields represent protein side chain ensembles?

PubMed

Petrović, Dušan; Wang, Xue; Strodel, Birgit

2018-05-23

Although the protein backbone is the most fundamental part of the structure, the fine-tuning of side-chain conformations is important for protein function, for example, in protein-protein and protein-ligand interactions, and also in enzyme catalysis. While several benchmarks testing the performance of protein force fields for side chain properties have already been published, they often considered only a few force fields and were not tested against the same experimental observables; hence, they are not directly comparable. In this work, we explore the ability of twelve force fields, which are different flavors of AMBER, CHARMM, OPLS, or GROMOS, to reproduce average rotamer angles and rotamer populations obtained from extensive NMR studies of the 3 J and residual dipolar coupling constants for two small proteins: ubiquitin and GB3. Based on a total of 196 μs sampling time, our results reveal that all force fields identify the correct side chain angles, while the AMBER and CHARMM force fields clearly outperform the OPLS and GROMOS force fields in estimating rotamer populations. The three best force fields for representing the protein side chain dynamics are AMBER 14SB, AMBER 99SB*-ILDN, and CHARMM36. Furthermore, we observe that the side chain ensembles of buried amino acid residues are generally more accurately represented than those of the surface exposed residues. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Perlinhibin, a Cysteine-, Histidine-, and Arginine-Rich Miniprotein from Abalone (Haliotis laevigata) Nacre, Inhibits In Vitro Calcium Carbonate Crystallization

PubMed Central

Mann, Karlheinz; Siedler, Frank; Treccani, Laura; Heinemann, Fabian; Fritz, Monika

2007-01-01

We have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata (greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids was determined by Edman degradation supported by mass spectrometry. The most abundant amino acids were cysteine (19.5%), histidine (17%), and arginine (14.6%). The positively charged amino acids were almost counterbalanced by negatively charged ones resulting in a calculated isoelectric point of 7.86. Atomic-force microscopy studies of the interaction of the protein with calcite surfaces in supersaturated calcium carbonate solution or calcium chloride solution showed that the protein bound specifically to calcite steps, inhibiting further crystal growth at these sites in carbonate solution and preventing crystal dissolution when carbonate was substituted with chloride. Therefore this protein was named perlinhibin. X-ray diffraction investigation of the crystal after atomic-force microscopy growth experiments showed that the formation of aragonite was induced on the calcite substrate around holes caused by perlinhibin crystal-growth inhibition. The strong interaction of the protein with calcium carbonate was also shown by vapor diffusion crystallization. In the presence of the protein, the crystal surfaces were covered with holes due to protein binding and local inhibition of crystal growth. In addition to perlinhibin, we isolated and sequenced a perlinhibin-related protein, indicating that perlinhibin may be a member of a family of closely related proteins. PMID:17496038

Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy.

PubMed

Laux, Eva-Maria; Knigge, Xenia; Bier, Frank F; Wenger, Christian; Hölzel, Ralph

2015-09-01

The combination of alternating electric fields with nanometer-sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real-time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The validity of an animal model for experiments related to weightlessness

NASA Technical Reports Server (NTRS)

Musacchia, X. J.; Steffen, J. M.

1983-01-01

Animal evolution has witnessed morphological and physiological adaptations to gravitational forces. In the rat, hind limb muscles can be used to illustrate a range of load bearing functions: soleus - gastrocnemius = plantaris - extensor digitorum longus (EDL). A harness suspension apparatus is used to induce hypokinesia and hypodynamia (H&H) and to simulate responses comparable to those seen in weightlessness (i.e., COSMOS experiments). After one and two weeks of suspension H&H, there is muscle atrophy with a loss in muscle mass; the result of loss in muscle protein. Concommitantly, there is a decrease in RNA, but not in DNA content. The effects are greatest in the soleus and least in the EDL. These recent findings, in concert with earlier reports of increased nitrogenous excretion, suggest that both decreased protein synthesis and increased protein catabolism are characteristic of muscle atrophy. Recovery is seen in terms of reversal of these effects after removal from suspension.

Molecular simulation of the effect of cholesterol on lipid-mediated protein-protein interactions.

PubMed

de Meyer, Frédérick J-M; Rodgers, Jocelyn M; Willems, Thomas F; Smit, Berend

2010-12-01

Experiments and molecular simulations have shown that the hydrophobic mismatch between proteins and membranes contributes significantly to lipid-mediated protein-protein interactions. In this article, we discuss the effect of cholesterol on lipid-mediated protein-protein interactions as function of hydrophobic mismatch, protein diameter and protein cluster size, lipid tail length, and temperature. To do so, we study a mesoscopic model of a hydrated bilayer containing lipids and cholesterol in which proteins are embedded, with a hybrid dissipative particle dynamics-Monte Carlo method. We propose a mechanism by which cholesterol affects protein interactions: protein-induced, cholesterol-enriched, or cholesterol-depleted lipid shells surrounding the proteins affect the lipid-mediated protein-protein interactions. Our calculations of the potential of mean force between proteins and protein clusters show that the addition of cholesterol dramatically reduces repulsive lipid-mediated interactions between proteins (protein clusters) with positive mismatch, but does not affect attractive interactions between proteins with negative mismatch. Cholesterol has only a modest effect on the repulsive interactions between proteins with different mismatch. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Coarse-grained Simulations of Sugar Transport and Conformational Changes of Lactose Permease

NASA Astrophysics Data System (ADS)

Liu, Jin; Jewel, S. M. Yead; Dutta, Prashanta

2016-11-01

Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H+ symport process. Lactose/H+ symport is a highly complex process that involves sugar translocation, H+ transfer, as well as large-scale protein conformational changes. The complete picture of lactose/H+ symport is largely unclear due to the complexity and multiscale nature of the process. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid. After validation, we implement the new force field to investigate the transport of a β-D-galactopyranosyl-1-thio- β-D-galactopyranoside (TDG) molecule across a wild-type LacY during lactose/H+ symport process. Results show that the local interactions between TDG and LacY at the binding pocket are consistent with the X-ray experiment. Protonation of Glu325 stabilizes the TDG and inward-facing conformation of LacY. Protonation of Glu269 induces a dramatic protein structural reorganization and causes the expulsion of TDG from LacY to both sides of the membrane. The structural changes occur primarily in the N-terminal domain of LacY. This work is supported by NSF Grants: CBET-1250107 and CBET -1604211.

Loss of electrostatic cell-surface repulsion mediates myelin membrane adhesion and compaction in the central nervous system.

PubMed

Bakhti, Mostafa; Snaidero, Nicolas; Schneider, David; Aggarwal, Shweta; Möbius, Wiebke; Janshoff, Andreas; Eckhardt, Matthias; Nave, Klaus-Armin; Simons, Mikael

2013-02-19

During the development of the central nervous system (CNS), oligodendrocytes wrap their plasma membrane around axons to form a multilayered stack of tightly attached membranes. Although intracellular myelin compaction and the role of myelin basic protein has been investigated, the forces that mediate the close interaction of myelin membranes at their external surfaces are poorly understood. Such extensive bilayer-bilayer interactions are usually prevented by repulsive forces generated by the glycocalyx, a dense and confluent layer of large and negatively charged oligosaccharides. Here we investigate the molecular mechanisms underlying myelin adhesion and compaction in the CNS. We revisit the role of the proteolipid protein and analyze the contribution of oligosaccharides using cellular assays, biophysical tools, and transgenic mice. We observe that differentiation of oligodendrocytes is accompanied by a striking down-regulation of components of their glycocalyx. Both in vitro and in vivo experiments indicate that the adhesive properties of the proteolipid protein, along with the reduction of sialic acid residues from the cell surface, orchestrate myelin membrane adhesion and compaction in the CNS. We suggest that loss of electrostatic cell-surface repulsion uncovers weak and unspecific attractive forces in the bilayer that bring the extracellular surfaces of a membrane into close contact over long distances.

Propensity to form amyloid fibrils is encoded as excitations in the free energy landscape of monomeric proteins.

PubMed

Zhuravlev, Pavel I; Reddy, Govardhan; Straub, John E; Thirumalai, D

2014-07-15

Protein aggregation, linked to many of diseases, is initiated when monomers access rogue conformations that are poised to form amyloid fibrils. We show, using simulations of src SH3 domain, that mechanical force enhances the population of the aggregation-prone (N(⁎)) states, which are rarely populated under force free native conditions but are encoded in the spectrum of native fluctuations. The folding phase diagrams of SH3 as a function of denaturant concentration ([C]), mechanical force (f), and temperature exhibit an apparent two-state behavior, without revealing the presence of the elusive N(⁎) states. Interestingly, the phase boundaries separating the folded and unfolded states at all [C] and f fall on a master curve, which can be quantitatively described using an analogy to superconductors in a magnetic field. The free energy profiles as a function of the molecular extension (R), which are accessible in pulling experiments, (R), reveal the presence of a native-like N(⁎) with a disordered solvent-exposed amino-terminal β-strand. The structure of the N(⁎) state is identical with that found in Fyn SH3 by NMR dispersion experiments. We show that the timescale for fibril formation can be estimated from the population of the N(⁎) state, determined by the free energy gap separating the native structure and the N(⁎) state, a finding that can be used to assess fibril forming tendencies of proteins. The structures of the N(⁎) state are used to show that oligomer formation and likely route to fibrils occur by a domain-swap mechanism in SH3 domain. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular dynamics and Monte Carlo simulations resolve apparent diffusion rate differences for proteins confined in nanochannels

DOE PAGES

Tringe, J. W.; Ileri, N.; Levie, H. W.; ...

2015-08-01

We use Molecular Dynamics and Monte Carlo simulations to examine molecular transport phenomena in nanochannels, explaining four orders of magnitude difference in wheat germ agglutinin (WGA) protein diffusion rates observed by fluorescence correlation spectroscopy (FCS) and by direct imaging of fluorescently-labeled proteins. We first use the ESPResSo Molecular Dynamics code to estimate the surface transport distance for neutral and charged proteins. We then employ a Monte Carlo model to calculate the paths of protein molecules on surfaces and in the bulk liquid transport medium. Our results show that the transport characteristics depend strongly on the degree of molecular surface coverage.more » Atomic force microscope characterization of surfaces exposed to WGA proteins for 1000 s show large protein aggregates consistent with the predicted coverage. These calculations and experiments provide useful insight into the details of molecular motion in confined geometries.« less

Asymmetrically localized proteins stabilize basal bodies against ciliary beating forces

PubMed Central

Galati, Domenico F.

2016-01-01

Basal bodies are radially symmetric, microtubule-rich structures that nucleate and anchor motile cilia. Ciliary beating produces asymmetric mechanical forces that are resisted by basal bodies. To resist these forces, distinct regions within the basal body ultrastructure and the microtubules themselves must be stable. However, the molecular components that stabilize basal bodies remain poorly defined. Here, we determine that Fop1 functionally interacts with the established basal body stability components Bld10 and Poc1. We find that Fop1 and microtubule glutamylation incorporate into basal bodies at distinct stages of assembly, culminating in their asymmetric enrichment at specific triplet microtubule regions that are predicted to experience the greatest mechanical force from ciliary beating. Both Fop1 and microtubule glutamylation are required to stabilize basal bodies against ciliary beating forces. Our studies reveal that microtubule glutamylation and Bld10, Poc1, and Fop1 stabilize basal bodies against the forces produced by ciliary beating via distinct yet interdependent mechanisms. PMID:27807131

The work of titin protein folding as a major driver in muscle contraction

PubMed Central

Eckels, Edward C.; Tapia-Rojo, Rafael; Rivas-Pardo, Jamie Andrés; Fernández, Julio M.

2018-01-01

Single molecule atomic force microscopy and magnetic tweezers experiments have demonstrated that titin Ig domains are capable of folding against a pulling force, generating mechanical work which exceeds that produced by a myosin motor. We hypothesize that upon muscle activation, formation of actomyosin crossbridges reduces the force on titin causing entropic recoil of the titin polymer and triggering the folding of the titin Ig domains. In the physiological force range of 4–15 pN under which titin operates in muscle, the folding contraction of a single Ig domain can generate 200% of the work of entropic recoil, and occurs at forces which exceed the maximum stalling force of single myosin motors. Thus titin operates like a mechanical battery storing elastic energy efficiently by unfolding Ig domains, and delivering the charge back by folding when the motors are activated during a contraction. We advance the hypothesis that titin folding and myosin activation act as inextricable partners during muscle contraction. PMID:29433413

Lipid14: The Amber Lipid Force Field

PubMed Central

2015-01-01

The AMBER lipid force field has been updated to create Lipid14, allowing tensionless simulation of a number of lipid types with the AMBER MD package. The modular nature of this force field allows numerous combinations of head and tail groups to create different lipid types, enabling the easy insertion of new lipid species. The Lennard-Jones and torsion parameters of both the head and tail groups have been revised and updated partial charges calculated. The force field has been validated by simulating bilayers of six different lipid types for a total of 0.5 μs each without applying a surface tension; with favorable comparison to experiment for properties such as area per lipid, volume per lipid, bilayer thickness, NMR order parameters, scattering data, and lipid lateral diffusion. As the derivation of this force field is consistent with the AMBER development philosophy, Lipid14 is compatible with the AMBER protein, nucleic acid, carbohydrate, and small molecule force fields. PMID:24803855

Novel nonlinear knowledge-based mean force potentials based on machine learning.

PubMed

Dong, Qiwen; Zhou, Shuigeng

2011-01-01

The prediction of 3D structures of proteins from amino acid sequences is one of the most challenging problems in molecular biology. An essential task for solving this problem with coarse-grained models is to deduce effective interaction potentials. The development and evaluation of new energy functions is critical to accurately modeling the properties of biological macromolecules. Knowledge-based mean force potentials are derived from statistical analysis of proteins of known structures. Current knowledge-based potentials are almost in the form of weighted linear sum of interaction pairs. In this study, a class of novel nonlinear knowledge-based mean force potentials is presented. The potential parameters are obtained by nonlinear classifiers, instead of relative frequencies of interaction pairs against a reference state or linear classifiers. The support vector machine is used to derive the potential parameters on data sets that contain both native structures and decoy structures. Five knowledge-based mean force Boltzmann-based or linear potentials are introduced and their corresponding nonlinear potentials are implemented. They are the DIH potential (single-body residue-level Boltzmann-based potential), the DFIRE-SCM potential (two-body residue-level Boltzmann-based potential), the FS potential (two-body atom-level Boltzmann-based potential), the HR potential (two-body residue-level linear potential), and the T32S3 potential (two-body atom-level linear potential). Experiments are performed on well-established decoy sets, including the LKF data set, the CASP7 data set, and the Decoys “R”Us data set. The evaluation metrics include the energy Z score and the ability of each potential to discriminate native structures from a set of decoy structures. Experimental results show that all nonlinear potentials significantly outperform the corresponding Boltzmann-based or linear potentials, and the proposed discriminative framework is effective in developing knowledge-based mean force potentials. The nonlinear potentials can be widely used for ab initio protein structure prediction, model quality assessment, protein docking, and other challenging problems in computational biology.

Mechanical Regulation in Cell Division and in Neurotransmitter Release

NASA Astrophysics Data System (ADS)

Thiyagarajan, Sathish

During their lifecycle, cells must produce forces which play important roles in several subcellular processes. Force-producing components are organized into macromolecular assemblies of proteins that are often dynamic, and are constructed or disassembled in response to various signals. The forces themselves may directly be involved in subcellular mechanics, or they may influence mechanosensing proteins either within or outside these structures. These proteins play different roles: they may ensure the stability of the force-producing structure, or they may send signals to a coupled process. The generation and sensing of subcellular forces is an active research topic, and this thesis focusses on the roles of these forces in two key areas: cell division and neurotransmitter release. The first part of the thesis deals with the effect of force on cell wall growth regulation during division in the fission yeast Schizosaccharomyces pombe, a cigar-shaped, unicellular organism. During cytokinesis, the last stage of cell division in which the cell physically divides into two, a tense cytokinetic ring anchored to the cellular membrane assembles and constricts, accompanied by the inward centripetal growth of new cell wall, called septum, in the wake of the inward-moving membrane. The contour of the septum hole maintains its circularity as it reduces in size--an indication of regulated growth. To characterize the cell wall growth process, we performed image analysis on contours of the leading edge of the septum obtained via fluorescence microscopy in the labs of our collaborators. We quantified the deviations from circularity using the edge roughness. The roughness was spatially correlated, suggestive of regulated growth. We hypothesized that the cell wall growers are mechanosensitive and respond to the force exerted by the ring. A mathematical model based on this hypothesis then showed that this leads to corrections of roughness in a curvature-dependent fashion. Thus, one of the roles of ring tension is to communicate with the mechanosensitive septum growth processes and coordinate growth to ensure the daughter cells have a functional cell wall. The second part of the thesis deals with how ring tension is produced and sustained, using experimentally measured ultrastructure of the cytokinetic ring itself. Recent super-resolution experiments have revealed that several key proteins of the fission yeast constricting ring are organized into membrane-anchored complexes called nodes. The force producing protein myosin-II in these nodes exerts pulling forces on polymeric actin filaments that are synthesized from polymerizers residing in the nodes. How these forces are marshalled to generate ring tension, and how such an organization maintains its stability is unclear. Using a mathematical model with coarse-grained representations of actin and myosin, we showed that such a node-based organization reproduces previously measured ring tension values. The model explains the origin of experimentally observed bidirectional motion of the nodes in the ring, and showed that turnover of the nodes rescues the ring from inherent contractile instabilities that would be expected when a force-producing structure is made up of small object that effectively attract one another. Finally, the third part of the thesis deals with the role of forces produced by SNARE proteins at synapses between two neurons during neurotransmission. A key step here is synaptic release, where inside a neuron, membrane-bound compartments called vesicles filled with neurotransmitter fuse with the membrane of the neuron forming a transient fusion pore, and release their contents to the outside of the cell. These neurotransmitter molecules are sensed by another neuron that is physically separate from the neuron in question and this neuron propagates the signal henceforth. Thus, regulation of neurotransmitter release is a key step in neurotransmission. A fusion machinery consisting of several proteins facilitates membrane fusion, and pore nucleation requires the formation of a SNARE protein complex in this machinery, whose role during pore dilation is unclear. Using electrophysiological measurements, our collaborators experimentally measured the statistics of the size of single fusion pores in vitro, and observed that average pore sizes increased with the number of SNARE proteins. Using mathematical modeling, we showed that this effect was due to an entropic crowding force that expands the pore and increases with the number of SNAREs, and counteracts the energy barrier to fusion pore expansion.

Nanoindentation studies of full and empty viral capsids and the effects of capsid protein mutations on elasticity and strength

PubMed Central

Michel, J. P.; Ivanovska, I. L.; Gibbons, M. M.; Klug, W. S.; Knobler, C. M.; Wuite, G. J. L.; Schmidt, C. F.

2006-01-01

The elastic properties of capsids of the cowpea chlorotic mottle virus have been examined at pH 4.8 by nanoindentation measurements with an atomic force microscope. Studies have been carried out on WT capsids, both empty and containing the RNA genome, and on full capsids of a salt-stable mutant and empty capsids of the subE mutant. Full capsids resisted indentation more than empty capsids, but all of the capsids were highly elastic. There was an initial reversible linear regime that persisted up to indentations varying between 20% and 30% of the diameter and applied forces of 0.6–1.0 nN; it was followed by a steep drop in force that is associated with irreversible deformation. A single point mutation in the capsid protein increased the capsid stiffness. The experiments are compared with calculations by finite element analysis of the deformation of a homogeneous elastic thick shell. These calculations capture the features of the reversible indentation region and allow Young's moduli and relative strengths to be estimated for the empty capsids. PMID:16606825

Surface force measurements and simulations of mussel-derived peptide adhesives on wet organic surfaces

PubMed Central

Levine, Zachary A.; Rapp, Michael V.; Wei, Wei; Mullen, Ryan Gotchy; Wu, Chun; Zerze, Gül H.; Mittal, Jeetain; Waite, J. Herbert; Israelachvili, Jacob N.; Shea, Joan-Emma

2016-01-01

Translating sticky biological molecules—such as mussel foot proteins (MFPs)—into synthetic, cost-effective underwater adhesives with adjustable nano- and macroscale characteristics requires an intimate understanding of the glue’s molecular interactions. To help facilitate the next generation of aqueous adhesives, we performed a combination of surface forces apparatus (SFA) measurements and replica-exchange molecular dynamics (REMD) simulations on a synthetic, easy to prepare, Dopa-containing peptide (MFP-3s peptide), which adheres to organic surfaces just as effectively as its wild-type protein analog. Experiments and simulations both show significant differences in peptide adsorption on CH3-terminated (hydrophobic) and OH-terminated (hydrophilic) self-assembled monolayers (SAMs), where adsorption is strongest on hydrophobic SAMs because of orientationally specific interactions with Dopa. Additional umbrella-sampling simulations yield free-energy profiles that quantitatively agree with SFA measurements and are used to extract the adhesive properties of individual amino acids within the context of MFP-3s peptide adhesion, revealing a delicate balance between van der Waals, hydrophobic, and electrostatic forces. PMID:27036002

Enhanced Dielectrophoretic Enrichment of Nanoparticles Using a Nanostructured Tip for Nanoengineered Medicine and Biology

NASA Astrophysics Data System (ADS)

Yeo, Woonhong

2011-12-01

Enrichment of low-concentration nanoparticles (NPs) is of great interest in the fields of medicine, biology, and environment. In particular, the enrichment of bioparticles such as virus, quantum dots, DNA, or protein can have broad impacts on disease diagnosis, drug discovery, and environmental monitoring. Currently available NP enrichment methods employ centrifugation, microfiltration, or magnetic field. However, these methods are limited in cumbersome preparation steps, low yield, and low throughput. Electric field-based methods have demonstrated potential for NP enrichment, but two-dimensional planar electrodes are limited in sensitivity, molecular transfer, and imaging capability. In addition, the detection of low abundance, non-amplifiable particles such as proteins and metals is very challenging due to the low efficiency of current methods. In this dissertation, the challenges are addressed by nanotip-based NP enrichment. Fundamentals of NP enrichment are studied with a nanostructured tip. The nanotip-based NP enrichment is investigated by correlating a dielectrophoretic (DEP) force with Brownian motion force. In experiment, the predicted NP enrichment is validated by using gold (Au) NPs. The DEP effective distance for NP enrichment with a nanotip is suggested. Sequence-specific enrichment of oligonucleotides is studied by considering DEP force, Brownian motion, and affinity binding. In experiment, the optimal parameters for ultimate enrichment performance are studied using a hybridization assay. In the assay, a nanotip is functionalized with probe-oligonucleotides for sequence-specific binding. Size-specific NP enrichment is explored by studying DEP, capillary action, and viscosity. The capillary action force with a nanotip is calculated analytically, which is then compared with the DEP force. The viscosity effect is considered for NP capturing on a nanotip. The studied size-specific enrichment mechanism is validated in experiment by using various polystyrene nanospheres. The studied enrichment mechanism of NPs with a nanotip is applied to the detection of viral particles. In the characterization study, T7 viral particles having 50 nm in diameter are observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). In experiment, the viral particles in a buffer are enriched to a nanotip by DEP, and captured onto the nanotip by DEP and viscosity. The captured viral particles on the nanotip are detected by fluorescence microscopy for whole nanotip observation, and validated by SEM. The enhanced DEP enrichment of NPs using a nanotip shows great potential for highly sensitive NP detection and analysis in nanoengineered medicine and biology.

Unfolding DNA condensates produced by DNA-like charged depletants: A force spectroscopy study

NASA Astrophysics Data System (ADS)

Lima, C. H. M.; Rocha, M. S.; Ramos, E. B.

2017-02-01

In this work, we have measured, by means of optical tweezers, forces acting on depletion-induced DNA condensates due to the presence of the DNA-like charged protein bovine serum albumin (BSA). The stretching and unfolding measurements performed on the semi-flexible DNA chain reveal (1) the softening of the uncondensed DNA contour length and (2) a mechanical behavior strikingly different from those previously observed: the force-extension curves of BSA-induced DNA condensates lack the "saw-tooth" pattern and applied external forces as high as ≈80 pN are unable to fully unfold the condensed DNA contour length. This last mechanical experimental finding is in agreement with force-induced "unpacking" detailed Langevin dynamics simulations recently performed by Cortini et al. on model rod-like shaped condensates. Furthermore, a simple thermodynamics analysis of the unfolding process has enabled us to estimate the free energy involved in the DNA condensation: the estimated depletion-induced interactions vary linearly with both the condensed DNA contour length and the BSA concentration, in agreement with the analytical and numerical analysis performed on model DNA condensates. We hope that future additional experiments can decide whether the rod-like morphology is the actual one we are dealing with (e.g. pulling experiments coupled with super-resolution fluorescence microscopy).

Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism.

PubMed

Riveline, D; Zamir, E; Balaban, N Q; Schwarz, U S; Ishizaki, T; Narumiya, S; Kam, Z; Geiger, B; Bershadsky, A D

2001-06-11

The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

PubMed

Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

2015-11-05

Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lateral fluid flow fractionation using dielectrophoresis (LFFF-DEP) for size-independent, label-free isolation of circulating tumor cells.

PubMed

Waheed, Waqas; Alazzam, Anas; Mathew, Bobby; Christoforou, Nicolas; Abu-Nada, Eiyad

2018-06-15

This short communication introduces a continuous-flow, dielectrophoresis-based lateral fluid flow fractionation microdevice for detection/isolation of circulating tumor cells in the presence of other haematological cells. The device utilizes two sets of planar interdigitated transducer electrodes micropatterned on top of a glass wafer using standard microfabrication techniques. A microchannel with a single inlet and two outlets, realized in polydimethylsiloxane, is bonded on the glass substrate. The two sets of electrodes slightly protrude into the microchannel. Both of the electrode sets are energized with signals at different frequencies and different operating voltages ensuring that the cancer cells experience positive dielectrophoretic force from one set of the electrodes and negative dielectrophoretic force from the other array. Normal cells experience unequal negative dielectrophoretic forces from opposing sets of electrodes. The resultant dielectrophoretic forces on cancer and normal cells push them to flow towards their designed outlets. Successful isolation of green fluorescent protein-labelled MDA-MB-231 breast cancer cells from regular blood cells, both suspended in a sucrose/dextrose medium, is reported in this work. Copyright © 2018 Elsevier B.V. All rights reserved.

Research objectives, opportunities, and facilities for microgravity science

NASA Technical Reports Server (NTRS)

Bayuzick, Robert J.

1992-01-01

Microgravity Science in the U.S.A. involves research in fluids science, combustion science, materials science, biotechnology, and fundamental physics. The purpose is to achieve a thorough understanding of the effects of gravitational body forces on physical phenomena relevant to those disciplines. This includes the study of phenomena which are usually overwhelmed by the presence of gravitational body forces and, therefore, chiefly manifested when gravitational forces are weak. In the pragmatic sense, the research involves gravity level as an experimental parameter. Calendar year 1992 is a landmark year for research opportunities in low earth orbit for Microgravity Science. For the first time ever, three Spacelab flights will fly in a single year: IML-1 was launched on January 22; USML-1 was launched on June 25; and, in September, SL-J will be launched. A separate flight involving two cargo bay carriers, USMP-1, will be launched in October. From the beginning of 1993 up to and including the Space Station era (1997), nine flights involving either Spacelab or USMP carriers will be flown. This will be augmented by a number of middeck payloads and get away specials flying on various flights. All of this activity sets the stage for experimentation on Space Station Freedom. Beginning in 1997, experiments in Microgravity Science will be conducted on the Space Station. Facilities for doing experiments in protein crystal growth, solidification, and biotechnology will all be available. These will be joined by middeck-class payloads and the microgravity glove box for conducting additional experiments. In 1998, a new generation protein crystal growth facility and a facility for conducting combustion research will arrive. A fluids science facility and additional capability for conducting research in solidification, as well as an ability to handle small payloads on a quick response basis, will be added in 1999. The year 2000 will see upgrades in the protein crystal growth and fluids science facilities. From the beginning of 1997 to the fall of 1999 (the 'man-tended capability' era), there will be two or three utilization flights per year. Plans call for operations in Microgravity Science during utilization flights and between utilization flights. Experiments conducted during utilization flights will characteristically require crew interaction, short duration, and less sensitivity to perturbations in the acceleration environment. Operations between utilization flights will involve experiments that can be controlled remotely and/or can be automated. Typically, the experiments will require long times and a pristine environment. Beyond the fall of 1999 (the 'permanently-manned capability' era), some payloads will require crew interaction; others will be automated and will make use of telescience.

Artificially-induced organelles are optimal targets for optical trapping experiments in living cells

PubMed Central

López-Quesada, C.; Fontaine, A.-S.; Farré, A.; Joseph, M.; Selva, J.; Egea, G.; Ludevid, M. D.; Martín-Badosa, E.; Montes-Usategui, M.

2014-01-01

Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads. PMID:25071944

Derivation of original RESP atomic partial charges for MD simulations of the LDAO surfactant with AMBER: applications to a model of micelle and a fragment of the lipid kinase PI4KA.

PubMed

Karakas, Esra; Taveneau, Cyntia; Bressanelli, Stéphane; Marchi, Massimo; Robert, Bruno; Abel, Stéphane

2017-01-01

In this paper, we describe the derivation and the validation of original RESP atomic partial charges for the N, N-dimethyl-dodecylamine oxide (LDAO) surfactant. These charges, designed to be fully compatible with all the AMBER force fields, are at first tested against molecular dynamics simulations of pure LDAO micelles and with a fragment of the lipid kinase PIK4A (DI) modeled with the QUARK molecular modeling server. To model the micelle, we used two distinct AMBER force fields (i.e. Amber99SB and Lipid14) and a variety of starting conditions. We find that the micelle structural properties (such as the shape, size, the LDAO headgroup hydration, and alkyl chain conformation) slightly depend on the force field but not on the starting conditions and more importantly are in good agreement with experiments and previous simulations. We also show that the Lipid14 force field should be used instead of the Amber99SB one to better reproduce the C(sp3)C(sp3)C(sp3)C(sp3) conformation in the surfactant alkyl chain. Concerning the simulations with LDAO-DI protein, we carried out different runs at two NaCl concentrations (i.e. 0 and 300 mM) to mimic, in the latter case, the experimental conditions. We notice a small dependence of the simulation results with the LDAO parameters and the salt concentration. However, we find that in the simulations, three out of four tryptophans of the DI protein are not accessible to water in agreement with our fluorescence spectroscopy experiments reported in the paper.

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

NASA Astrophysics Data System (ADS)

Arnal, L.; Longo, G.; Stupar, P.; Castez, M. F.; Cattelan, N.; Salvarezza, R. C.; Yantorno, O. M.; Kasas, S.; Vela, M. E.

2015-10-01

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04644k

DNA Scrunching in the Packaging of Viral Genomes.

PubMed

Waters, James T; Kim, Harold D; Gumbart, James C; Lu, Xiang-Jun; Harvey, Stephen C

2016-07-07

The motors that drive double-stranded DNA (dsDNA) genomes into viral capsids are among the strongest of all biological motors for which forces have been measured, but it is not known how they generate force. We previously proposed that the DNA is not a passive substrate but that it plays an active role in force generation. This "scrunchworm hypothesis" holds that the motor proteins repeatedly dehydrate and rehydrate the DNA, which then undergoes cyclic shortening and lengthening motions. These are captured by a coupled protein-DNA grip-and-release cycle to rectify the motion and translocate the DNA into the capsid. In this study, we examined the interactions of dsDNA with the dodecameric connector protein of bacteriophage ϕ29, using molecular dynamics simulations on four different DNA sequences, starting from two different conformations (A-DNA and B-DNA). In all four simulations starting with the protein equilibrated with A-DNA in the channel, we observed transitions to a common, metastable, highly scrunched conformation, designated A*. This conformation is very similar to one recently reported by Kumar and Grubmüller in much longer MD simulations on B-DNA docked into the ϕ29 connector. These results are significant for four reasons. First, the scrunched conformations occur spontaneously, without requiring lever-like protein motions often believed to be necessary for DNA translocation. Second, the transition takes place within the connector, providing the location of the putative "dehydrator". Third, the protein has more contacts with one strand of the DNA than with the other; the former was identified in single-molecule laser tweezer experiments as the "load-bearing strand". Finally, the spontaneity of the DNA-protein interaction suggests that it may play a role in the initial docking of DNA in motors like that of T4 that can load and package any sequence.

Force-extension behavior of DNA in the presence of DNA-bending nucleoid associated proteins

NASA Astrophysics Data System (ADS)

Dahlke, K.; Sing, C. E.

2018-02-01

Interactions between nucleoid associated proteins (NAPs) and DNA affect DNA polymer conformation, leading to phenomena such as concentration dependent force-extension behavior. These effects, in turn, also impact the local binding behavior of the protein, such as high forces causing proteins to unbind, or proteins binding favorably to locally bent DNA. We develop a coarse-grained NAP-DNA simulation model that incorporates both force- and concentration-dependent behaviors, in order to study the interplay between NAP binding and DNA conformation. This model system includes multi-state protein binding and unbinding, motivated by prior work, but is now dependent on the local structure of the DNA, which is related to external forces acting on the DNA strand. We observe the expected qualitative binding behavior, where more proteins are bound at lower forces than at higher forces. Our model also includes NAP-induced DNA bending, which affects DNA elasticity. We see semi-quantitative matching of our simulated force-extension behavior to the reported experimental data. By using a coarse-grained simulation, we are also able to look at non-equilibrium behaviors, such as dynamic extension of a DNA strand. We stretch a DNA strand at different rates and at different NAP concentrations to observe how the time scales of the system (such as pulling time and unbinding time) work in concert. When these time scales are similar, we observe measurable rate-dependent changes in the system, which include the number of proteins bound and the force required to extend the DNA molecule. This suggests that the relative time scales of different dynamic processes play an important role in the behavior of NAP-DNA systems.

Novel Applications of Magnetic Fields for Fluid Flow Control and for Simulating Variable Gravity Conditions

NASA Technical Reports Server (NTRS)

Ramachandran, N.

2005-01-01

Static and dynamic magnetic fields have been used to control convection in many materials processing applications. In most of the applications, convection control (damping or enhancement) is achieved through the Lorentz force that can be tailored to counteract/assist dominant system flows. This technique has been successfully applied to liquids that are electrically conducting, such as high temperature melts of semiconductors, metals and alloys, etc. In liquids with low electrical conductivity such as ionic solutions of salts in water, the Lorentz force is weak and hence not very effective and alternate ways of flow control are necessary. If the salt in solution is paramagnetic then the variation of magnetic susceptibility with temperature and/or concentration can be used for flow control. For thermal buoyancy driven flows this can be accomplished in a temperature range below the Curie point of the salt. The magnetic force is proportional to the magnetic susceptibility and the product of the magnetic field and its gradient. By suitably positioning the experiment cell in the magnet, system flows can be assisted or countered, as desired. A similar approach can be extended to diamagnetic substances and fluids but the required magnetic force is considerably larger than that required for paramagnetic substances. The presentation will provide an overview of work to date on a NASA fluid physics sponsored project that aims to test the hypothesis of convective flow control using strong magnetic fields in protein crystal growth. The objective is to understand the nature of the various forces that come into play, delineate causative factors for fluid flow and to quantify them through experiments, analysis, and numerical modeling. The seminar will report specifically on the experimental results using paramagnetic salts and solutions in magnetic fields and compare them to analytical predictions. Applications of the concept to protein crystallization studies will be discussed. The use of strong magnetic fields for terrestrially simulating variable gravity environments and applications supporting the NASA Exploration Initiative will also be briefly discussed.

Molecular simulations of lipid-mediated protein-protein interactions.

PubMed

de Meyer, Frédérick Jean-Marie; Venturoli, Maddalena; Smit, Berend

2008-08-01

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.

Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction.

PubMed

Sotomayor, Marcos; Weihofen, Wilhelm A; Gaudet, Rachelle; Corey, David P

2012-12-06

Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals. Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links, fine filaments formed by two atypical cadherins known as protocadherin 15 and cadherin 23 (refs 4, 5). These two proteins are involved in inherited deafness and feature long extracellular domains that interact tip-to-tip in a Ca(2+)-dependent manner. However, the molecular architecture of this complex is unknown. Here we combine crystallography, molecular dynamics simulations and binding experiments to characterize the protocadherin 15-cadherin 23 bond. We find a unique cadherin interaction mechanism, in which the two most amino-terminal cadherin repeats (extracellular cadherin repeats 1 and 2) of each protein interact to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex is shown to become unstable in response to Ca(2+) removal owing to increased flexure of Ca(2+)-free cadherin repeats. Finally, we use structures and biochemical measurements to study the molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis, neural connectivity and cancer.

Single molecule force measurements delineate salt, pH and surface effects on biopolymer adhesion

NASA Astrophysics Data System (ADS)

Pirzer, T.; Geisler, M.; Scheibel, T.; Hugel, T.

2009-06-01

In this paper we probe the influence of surface properties, pH and salt on the adhesion of recombinant spider silk proteins onto solid substrates with single molecule force spectroscopy. A single engineered spider silk protein (monomeric C16 or dimeric (QAQ)8NR3) is covalently bound with one end to an AFM tip, which assures long-time measurements for hours with one and the same protein. The tip with the protein is brought into contact with various substrates at various buffer conditions and then retracted to desorb the protein. We observe a linear dependence of the adhesion force on the concentration of three selected salts (NaCl, NaH2PO4 and NaI) and a Hofmeister series both for anions and cations. As expected, the more hydrophobic C16 shows a higher adhesion force than (QAQ)8NR3, and the adhesion force rises with the hydrophobicity of the substrate. Unexpected is the magnitude of the dependences—we never observe a change of more than 30%, suggesting a surprisingly well-regulated balance between dispersive forces, water-structure-induced forces as well as co-solute-induced forces in biopolymer adhesion.

Quantifying Young's moduli of protein fibrils and particles with bimodal force spectroscopy.

PubMed

Gilbert, Jay; Charnley, Mirren; Cheng, Christopher; Reynolds, Nicholas P; Jones, Owen G

2017-10-19

Force spectroscopy is a means of obtaining mechanical information of individual nanometer-scale structures in composite materials, such as protein assemblies for use in consumer films or gels. As a recently developed force spectroscopy technique, bimodal force spectroscopy relates frequency shifts in cantilevers simultaneously excited at multiple frequencies to the elastic properties of the contacted material, yet its utility for quantitative characterization of biopolymer assemblies has been limited. In this study, a linear correlation between experimental frequency shift and Young's modulus of polymer films was used to calibrate bimodal force spectroscopy and quantify Young's modulus of two protein nanostructures: β-lactoglobulin fibrils and zein nanoparticles. Cross-sectional Young's modulus of protein fibrils was determined to be 1.6 GPa while the modulus of zein nanoparticles was determined as 854 MPa. Parallel measurement of β-lactoglobulin fibril by a competing pulsed-force technique found a higher cross-sectional Young's modulus, highlighting the importance of comparative calibration against known standards in both pulsed and bimodal force spectroscopies. These findings demonstrate a successful procedure for measuring mechanical properties of individual protein assemblies with potential use in biological or packaging applications using bimodal force spectroscopy.

Stiffened lipid platforms at molecular force foci.

PubMed

Anishkin, Andriy; Kung, Ching

2013-03-26

How mechanical forces are sensed remains largely mysterious. The forces that gate prokaryotic and several eukaryotic channels were found to come from the lipid membrane. Our survey of animal cells found that membrane force foci all have cholesterol-gathering proteins and are reinforced with cholesterol. This result is evident in overt force sensors at the tips of stereocilia for vertebrate hearing and the touch receptor of Caenorhabditis elegans and mammalian neurons. For less specialized cells, cadherins sustain the force between neighboring cells and integrins between cells and matrix. These tension bearers also pass through and bind to a cholesterol-enriched platform before anchoring to cytoskeleton through other proteins. Cholesterol, in alliance with sphingomyelin and specialized proteins, enforces a more ordered structure in the bilayer. Such a stiffened platform can suppress mechanical noise, redirect, rescale, and confine force. We speculate that such platforms may be dynamic. The applied force may allow disordered-phase lipids to enter the platform-staging channel opening in the thinner mobile neighborhood. The platform may also contain specialized protein/lipid subdomains enclosing mechanosensitive channels to open with localized tension. Such a dynamic stage can mechanically operate structurally disparate channels or enzymes without having to tie them directly to cadherin, integrin, or other protein tethers.

Electrostatics of proteins in dielectric solvent continua. I. Newton's third law marries qE forces

NASA Astrophysics Data System (ADS)

Stork, Martina; Tavan, Paul

2007-04-01

The authors reformulate and revise an electrostatic theory treating proteins surrounded by dielectric solvent continua [B. Egwolf and P. Tavan, J. Chem. Phys. 118, 2039 (2003)] to make the resulting reaction field (RF) forces compatible with Newton's third law. Such a compatibility is required for their use in molecular dynamics (MD) simulations, in which the proteins are modeled by all-atom molecular mechanics force fields. According to the original theory the RF forces, which are due to the electric field generated by the solvent polarization and act on the partial charges of a protein, i.e., the so-called qE forces, can be quite accurately computed from Gaussian RF dipoles localized at the protein atoms. Using a slightly different approximation scheme also the RF energies of given protein configurations are obtained. However, because the qE forces do not account for the dielectric boundary pressure exerted by the solvent continuum on the protein, they do not obey the principle that actio equals reactio as required by Newton's third law. Therefore, their use in MD simulations is severely hampered. An analysis of the original theory has led the authors now to a reformulation removing the main difficulties. By considering the RF energy, which represents the dominant electrostatic contribution to the free energy of solvation for a given protein configuration, they show that its negative configurational gradient yields mean RF forces obeying the reactio principle. Because the evaluation of these mean forces is computationally much more demanding than that of the qE forces, they derive a suggestion how the qE forces can be modified to obey Newton's third law. Various properties of the thus established theory, particularly issues of accuracy and of computational efficiency, are discussed. A sample application to a MD simulation of a peptide in solution is described in the following paper [M. Stork and P. Tavan, J. Chem. Phys., 126, 165106 (2007).

Mechanical unfolding of an ankyrin repeat protein.

PubMed

Serquera, David; Lee, Whasil; Settanni, Giovanni; Marszalek, Piotr E; Paci, Emanuele; Itzhaki, Laura S

2010-04-07

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly alpha-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

i3Drefine Software for Protein 3D Structure Refinement and Its Assessment in CASP10

PubMed Central

Bhattacharya, Debswapna; Cheng, Jianlin

2013-01-01

Protein structure refinement refers to the process of improving the qualities of protein structures during structure modeling processes to bring them closer to their native states. Structure refinement has been drawing increasing attention in the community-wide Critical Assessment of techniques for Protein Structure prediction (CASP) experiments since its addition in 8th CASP experiment. During the 9th and recently concluded 10th CASP experiments, a consistent growth in number of refinement targets and participating groups has been witnessed. Yet, protein structure refinement still remains a largely unsolved problem with majority of participating groups in CASP refinement category failed to consistently improve the quality of structures issued for refinement. In order to alleviate this need, we developed a completely automated and computationally efficient protein 3D structure refinement method, i3Drefine, based on an iterative and highly convergent energy minimization algorithm with a powerful all-atom composite physics and knowledge-based force fields and hydrogen bonding (HB) network optimization technique. In the recent community-wide blind experiment, CASP10, i3Drefine (as ‘MULTICOM-CONSTRUCT’) was ranked as the best method in the server section as per the official assessment of CASP10 experiment. Here we provide the community with free access to i3Drefine software and systematically analyse the performance of i3Drefine in strict blind mode on the refinement targets issued in CASP10 refinement category and compare with other state-of-the-art refinement methods participating in CASP10. Our analysis demonstrates that i3Drefine is only fully-automated server participating in CASP10 exhibiting consistent improvement over the initial structures in both global and local structural quality metrics. Executable version of i3Drefine is freely available at http://protein.rnet.missouri.edu/i3drefine/. PMID:23894517

Toward the description of electrostatic interactions between globular proteins: potential of mean force in the primitive model.

PubMed

Dahirel, Vincent; Jardat, Marie; Dufrêche, Jean-François; Turq, Pierre

2007-09-07

Monte Carlo simulations are used to calculate the exact potential of mean force between charged globular proteins in aqueous solution. The aim of the present paper is to study the influence of the ions of the added salt on the effective interaction between these nanoparticles. The charges of the model proteins, either identical or opposite, are either central or distributed on a discrete pattern. Contrarily to Poisson-Boltzmann predictions, attractive, and repulsive direct forces between proteins are not screened similarly. Moreover, it has been shown that the relative orientations of the charge patterns strongly influence salt-mediated interactions. More precisely, for short distances between the proteins, ions enhance the difference of the effective forces between (i) like-charged and oppositely charged proteins, (ii) attractive and repulsive relative orientations of the proteins, which may affect the selectivity of protein/protein recognition. Finally, such results observed with the simplest models are applied to a more elaborate one to demonstrate their generality.

Effects of surface wettability and contact time on protein adhesion to biomaterial surfaces

PubMed Central

Xu, Li-Chong; Siedlecki, Christopher A.

2013-01-01

Atomic force microscopy (AFM) was used to directly measure the adhesion forces between three test proteins and low density polyethylene (LDPE) surfaces treated by glow discharge plasma to yield various levels of water wettability. The adhesion of proteins to the LDPE substrates showed a step dependence on the wettability of surfaces as measured by the water contact angle (θ). For LDPE surfaces with θ > ∼60–65°, stronger adhesion forces were observed for bovine serum albumin, fibrinogen and human FXII than for the surfaces with θ < 60°. Smaller adhesion forces were observed for FXII than for the other two proteins on all surfaces although trends were identical. Increasing the contact time from 0 to 50 s for each protein–surface combination increased the adhesion force regardless of surface wettability. Time varying adhesion data was fit to an exponential model and free energies of protein unfolding were calculated. This data, viewed in light of previously published studies, suggests a 2-step model of protein denaturation, an early stage on the order of seconds to minutes where the outer surface of the protein interacts with the substrate and a second stage involving movement of hydrophobic amino acids from the protein core to the protein/surface interface. Impact statement The work described in this manuscript shows a stark transition between protein adherent and protein non-adherent materials in the range of water contact angles 60–65°, consistent with known changes in protein adsorption and activity. Time-dependent changes in adhesion force were used to calculate unfolding energies relating to protein–surface interactions. This analysis provides justification for a 2-step model of protein denaturation on surfaces. PMID:17466368

My 65 years in protein chemistry.

PubMed

Scheraga, Harold A

2015-05-01

This is a tour of a physical chemist through 65 years of protein chemistry from the time when emphasis was placed on the determination of the size and shape of the protein molecule as a colloidal particle, with an early breakthrough by James Sumner, followed by Linus Pauling and Fred Sanger, that a protein was a real molecule, albeit a macromolecule. It deals with the recognition of the nature and importance of hydrogen bonds and hydrophobic interactions in determining the structure, properties, and biological function of proteins until the present acquisition of an understanding of the structure, thermodynamics, and folding pathways from a linear array of amino acids to a biological entity. Along the way, with a combination of experiment and theoretical interpretation, a mechanism was elucidated for the thrombin-induced conversion of fibrinogen to a fibrin blood clot and for the oxidative-folding pathways of ribonuclease A. Before the atomic structure of a protein molecule was determined by x-ray diffraction or nuclear magnetic resonance spectroscopy, experimental studies of the fundamental interactions underlying protein structure led to several distance constraints which motivated the theoretical approach to determine protein structure, and culminated in the Empirical Conformational Energy Program for Peptides (ECEPP), an all-atom force field, with which the structures of fibrous collagen-like proteins and the 46-residue globular staphylococcal protein A were determined. To undertake the study of larger globular proteins, a physics-based coarse-grained UNited-RESidue (UNRES) force field was developed, and applied to the protein-folding problem in terms of structure, thermodynamics, dynamics, and folding pathways. Initially, single-chain and, ultimately, multiple-chain proteins were examined, and the methodology was extended to protein-protein interactions and to nucleic acids and to protein-nucleic acid interactions. The ultimate results led to an understanding of a variety of biological processes underlying natural and disease phenomena.

Biomolecularmodeling and simulation: a field coming of age

PubMed Central

Schlick, Tamar; Collepardo-Guevara, Rosana; Halvorsen, Leif Arthur; Jung, Segun; Xiao, Xia

2013-01-01

We assess the progress in biomolecular modeling and simulation, focusing on structure prediction and dynamics, by presenting the field’s history, metrics for its rise in popularity, early expressed expectations, and current significant applications. The increases in computational power combined with improvements in algorithms and force fields have led to considerable success, especially in protein folding, specificity of ligand/biomolecule interactions, and interpretation of complex experimental phenomena (e.g. NMR relaxation, protein-folding kinetics and multiple conformational states) through the generation of structural hypotheses and pathway mechanisms. Although far from a general automated tool, structure prediction is notable for proteins and RNA that preceded the experiment, especially by knowledge-based approaches. Thus, despite early unrealistic expectations and the realization that computer technology alone will not quickly bridge the gap between experimental and theoretical time frames, ongoing improvements to enhance the accuracy and scope of modeling and simulation are propelling the field onto a productive trajectory to become full partner with experiment and a field on its own right. PMID:21226976

Fiber networks amplify active stress

NASA Astrophysics Data System (ADS)

Lenz, Martin; Ronceray, Pierre; Broedersz, Chase

Large-scale force generation is essential for biological functions such as cell motility, embryonic development, and muscle contraction. In these processes, forces generated at the molecular level by motor proteins are transmitted by disordered fiber networks, resulting in large-scale active stresses. While fiber networks are well characterized macroscopically, this stress generation by microscopic active units is not well understood. I will present a comprehensive theoretical study of force transmission in these networks. I will show that the linear, small-force response of the networks is remarkably simple, as the macroscopic active stress depends only on the geometry of the force-exerting unit. In contrast, as non-linear buckling occurs around these units, local active forces are rectified towards isotropic contraction and strongly amplified. This stress amplification is reinforced by the networks' disordered nature, but saturates for high densities of active units. I will show that our predictions are quantitatively consistent with experiments on reconstituted tissues and actomyosin networks, and that they shed light on the role of the network microstructure in shaping active stresses in cells and tissue.

PACSAB: Coarse-Grained Force Field for the Study of Protein-Protein Interactions and Conformational Sampling in Multiprotein Systems.

PubMed

Emperador, Agustí; Sfriso, Pedro; Villarreal, Marcos Ariel; Gelpí, Josep Lluis; Orozco, Modesto

2015-12-08

Molecular dynamics simulations of proteins are usually performed on a single molecule, and coarse-grained protein models are calibrated using single-molecule simulations, therefore ignoring intermolecular interactions. We present here a new coarse-grained force field for the study of many protein systems. The force field, which is implemented in the context of the discrete molecular dynamics algorithm, is able to reproduce the properties of folded and unfolded proteins, in both isolation, complexed forming well-defined quaternary structures, or aggregated, thanks to its proper evaluation of protein-protein interactions. The accuracy and computational efficiency of the method makes it a universal tool for the study of the structure, dynamics, and association/dissociation of proteins.

Refolding dynamics of stretched biopolymers upon force quench

PubMed Central

Hyeon, Changbong; Morrison, Greg; Pincus, David L.; Thirumalai, D.

2009-01-01

Single-molecule force spectroscopy methods can be used to generate folding trajectories of biopolymers from arbitrary regions of the folding landscape. We illustrate the complexity of the folding kinetics and generic aspects of the collapse of RNA and proteins upon force quench by using simulations of an RNA hairpin and theory based on the de Gennes model for homopolymer collapse. The folding time, τF, depends asymmetrically on δfS = f S − f m and δf Q = f m − f Q where f S (f Q) is the stretch (quench) force and f m is the transition midforce of the RNA hairpin. In accord with experiments, the relaxation kinetics of the molecular extension, R(t), occurs in three stages: A rapid initial decrease in the extension is followed by a plateau and finally, an abrupt reduction in R(t) occurs as the native state is approached. The duration of the plateau increases as λ = τ Q/τ F decreases (where τ Q is the time in which the force is reduced from f S to f Q). Variations in the mechanisms of force-quench relaxation as λ is altered are reflected in the experimentally measurable time-dependent entropy, which is computed directly from the folding trajectories. An analytical solution of the de Gennes model under tension reproduces the multistage stage kinetics in R(t). The prediction that the initial stages of collapse should also be a generic feature of polymers is validated by simulation of the kinetics of toroid (globule) formation in semiflexible (flexible) homopolymers in poor solvents upon quenching the force from a fully stretched state. Our findings give a unified explanation for multiple disparate experimental observations of protein folding. PMID:19915145

Conformational changes and metastable states induced in proteins by green light

NASA Astrophysics Data System (ADS)

Comorosan, Sorin; Popescu, Irinel; Polosan, Silviu; Pirvu, Cristian; Ionescu, Elena; Paslaru, Liliana; Apostol, Marian

2015-01-01

In this paper we report conformational changes recorded on a protein molecule (α-amylase) under green light irradiation. In order to explain the experimental results we advanced the hypothesis that green light induces electric dipoles in the protein, which interact with each other, generating conformational modifications toward a more compact design, with different physical properties. The experiments were carried out with un-polarized light (λ = 520 nm) from a light-emitting-diode (1000 lm, 20 W, 105 mW on the target). In view of the character of our hypothesis, and corroborated with all our experimental results, we suggest that this phenomenon may be more extended and general, specific for a larger class of proteins, occurring on the protein macromolecules under the green light. The effects of α-amylase protein irradiation were revealed by circular dichroism, fluorescence, Raman and FTIR-spectroscopies, zeta potential, cyclic voltammetry, electric impedance spectroscopy and atomic force microscopy. Tentatively, we term the novel conformations as P∗ (polarized) proteins.

Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.

PubMed

Carrion-Vazquez, M; Oberhauser, A F; Fisher, T E; Marszalek, P E; Li, H; Fernandez, J M

2000-01-01

Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).

Bioassays Based on Molecular Nanomechanics

DOE PAGES

Majumdar, Arun

2002-01-01

Recent experiments have shown that when specific biomolecular interactions are confined to one surface of a microcantilever beam, changes in intermolecular nanomechanical forces provide sufficient differential torque to bend the cantilever beam. This has been used to detect single base pair mismatches during DNA hybridization, as well as prostate specific antigen (PSA) at concentrations and conditions that are clinically relevant for prostate cancer diagnosis. Since cantilever motion originates from free energy change induced by specific biomolecular binding, this technique is now offering a common platform for label-free quantitative analysis of protein-protein binding, DNA hybridization DNA-protein interactions, and in general receptor-ligandmore » interactions. Current work is focused on developing “universal microarrays” of microcantilever beams for high-throughput multiplexed bioassays.« less

Intrinsically Disordered Titin PEVK as a Molecular Velcro: Salt-Bridge Dynamics and Elasticity

NASA Astrophysics Data System (ADS)

Forbes, Jeffrey; Tsai, Wanxia; Wittebort, Richard; Wang, Kuan

2009-03-01

Titin is a giant modular protein (3-4 MDa) found in the muscle sarcomere, where the intrinsically disordered and elastic PEVK segment plays a major role in the passive tension of skeletal and heart tissues. We have proposed that salt-bridges play a central role in the elasticity of PEVK. The 50 kDa engineered PEVK polyprotein shows well-resolved NMR spectra at all concentrations. From long-range NOE's, we observed stable K to E salt-bridges. Simulated annealing with NMR restraints yielded a manifold of structures for an exon 172 trimer. Steered molecular dynamics simulations were done to study how the manifold of salt-bridges evolves during the stretching experiment. Repeated SMD simulations at slow velocity (0.0005 nm/ps) showed force spectra consistent with experimental AFM force spectra of the polyprotein. SMD shows that salt-bridges occur even at high degrees of stretch and that these short range interactions are in integral part of the mechanical properties of PEVK. We propose that the long-range, non-stereospecific nature of electrostatic interactions provide a facile mechanism to tether and untether the flexible chains, which in turn affect elasticity as well as control the accessibility of protein-protein interaction to these nanogel-like proteins.

Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1.

PubMed

Heidarsson, Pétur O; Naqvi, Mohsin M; Otazo, Mariela R; Mossa, Alessandro; Kragelund, Birthe B; Cecconi, Ciro

2014-09-09

Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.

GroEL stimulates protein folding through forced unfolding

PubMed Central

Lin, Zong; Madan, Damian; Rye, Hays S

2013-01-01

Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL’s physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding. PMID:18311152

Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample

NASA Astrophysics Data System (ADS)

Guo, Shifeng; Zhu, Xiaoying; Jańczewski, Dominik; Lee, Serina Siew Chen; He, Tao; Teo, Serena Lay Ming; Vancso, G. Julius

2016-09-01

Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the ‘footprint’ of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

Nanoscale observation of local bound charges of patterned protein arrays by scanning force microscopy

NASA Astrophysics Data System (ADS)

Oh, Y. J.; Jo, W.; Kim, S.; Park, S.; Kim, Y. S.

2008-09-01

A protein patterned surface using micro-contact printing methods has been investigated by scanning force microscopy. Electrostatic force microscopy (EFM) was utilized for imaging the topography and detecting the electrical properties such as the local bound charge distribution of the patterned proteins. It was found that the patterned IgG proteins are arranged down to 1 µm, and the 90° rotation of patterned anti-IgG proteins was successfully undertaken. Through the estimation of the effective areas, it was possible to determine the local bound charges of patterned proteins which have opposite electrostatic force behaviors. Moreover, we studied the binding probability between IgG and anti-IgG in a 1 µm2 MIMIC system by topographic and electrostatic signals for applicable label-free detections. We showed that the patterned proteins can be used for immunoassay of proteins on the functional substrate, and that they can also be used for bioelectronics device application, indicating distinct advantages with regard to accuracy and a label-free detection.

My 65 years in protein chemistry

PubMed Central

Scheraga, Harold A.

2015-01-01

This is a tour of a physical chemist through 65 years of protein chemistry from the time when emphasis was placed on the determination of the size and shape of the protein molecule as a colloidal particle, with an early breakthrough by James Sumner, followed by Linus Pauling and Fred Sanger, that a protein was a real molecule, albeit a macromolecule. It deals with the recognition of the nature and importance of hydrogen bonds and hydrophobic interactions in determining the structure, properties, and biological function of proteins until the present acquisition of an understanding of the structure, thermodynamics, and folding pathways from a linear array of amino acids to a biological entity. Along the way, with a combination of experiment and theoretical interpretation, a mechanism was elucidated for the thrombin-induced conversion of fibrinogen to a fibrin blood clot and for the oxidative-folding pathways of ribonuclease A. Before the atomic structure of a protein molecule was determined by x-ray diffraction or nuclear magnetic resonance spectroscopy, experimental studies of the fundamental interactions underlying protein structure led to several distance constraints which motivated the theoretical approach to determine protein structure, and culminated in the Empirical Conformational Energy Program for Peptides (ECEPP), an all-atom force field, with which the structures of fibrous collagen-like proteins and the 46-residue globular staphylococcal protein A were determined. To undertake the study of larger globular proteins, a physics-based coarse-grained UNited-RESidue (UNRES) force field was developed, and applied to the protein-folding problem in terms of structure, thermodynamics, dynamics, and folding pathways. Initially, single-chain and, ultimately, multiple-chain proteins were examined, and the methodology was extended to protein–protein interactions and to nucleic acids and to protein–nucleic acid interactions. The ultimate results led to an understanding of a variety of biological processes underlying natural and disease phenomena. PMID:25850343

Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus

NASA Astrophysics Data System (ADS)

von Bilderling, Catalina; Caldarola, Martín; Masip, Martín E.; Bragas, Andrea V.; Pietrasanta, Lía I.

2017-01-01

The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.

Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus.

PubMed

von Bilderling, Catalina; Caldarola, Martín; Masip, Martín E; Bragas, Andrea V; Pietrasanta, Lía I

2017-01-01

The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.

Stiffened lipid platforms at molecular force foci

PubMed Central

Anishkin, Andriy; Kung, Ching

2013-01-01

How mechanical forces are sensed remains largely mysterious. The forces that gate prokaryotic and several eukaryotic channels were found to come from the lipid membrane. Our survey of animal cells found that membrane force foci all have cholesterol-gathering proteins and are reinforced with cholesterol. This result is evident in overt force sensors at the tips of stereocilia for vertebrate hearing and the touch receptor of Caenorhabditis elegans and mammalian neurons. For less specialized cells, cadherins sustain the force between neighboring cells and integrins between cells and matrix. These tension bearers also pass through and bind to a cholesterol-enriched platform before anchoring to cytoskeleton through other proteins. Cholesterol, in alliance with sphingomyelin and specialized proteins, enforces a more ordered structure in the bilayer. Such a stiffened platform can suppress mechanical noise, redirect, rescale, and confine force. We speculate that such platforms may be dynamic. The applied force may allow disordered-phase lipids to enter the platform-staging channel opening in the thinner mobile neighborhood. The platform may also contain specialized protein/lipid subdomains enclosing mechanosensitive channels to open with localized tension. Such a dynamic stage can mechanically operate structurally disparate channels or enzymes without having to tie them directly to cadherin, integrin, or other protein tethers. PMID:23476066

High Tensile Strength of Engineered β-Solenoid Fibrils via Sonication and Pulling.

PubMed

Peng, Zeyu; Parker, Amanda S; Peralta, Maria D R; Ravikumar, Krishnakumar M; Cox, Daniel L; Toney, Michael D

2017-11-07

We present estimates of ultimate tensile strength (UTS) for two engineered β-solenoid protein mutant fibril structures (spruce budworm and Rhagium inquisitor antifreeze proteins) derived from sonication-based measurements and from force pulling molecular dynamics simulations, both in water. Sonication experiments generate limiting scissioned fibrils with a well-defined length-to-width correlation for the mutant spruce budworm protein and the resultant UTS estimate is 0.66 ± 0.08 GPa. For fibrils formed from engineered R. inquisitor antifreeze protein, depending upon geometry, we estimate UTSs of 3.5 ± 3.2-5.5 ± 5.1 GPa for proteins with interfacial disulfide bonds, and 1.6 ± 1.5-2.5 ± 2.3 GPa for the reduced form. The large error bars for the R. inquisitor structures are intrinsic to the broad distribution of limiting scission lengths. Simulations provide pulling velocity-dependent UTSs increasing from 0.2 to 1 GPa in the available speed range, and 1.5 GPa extrapolated to the speeds expected in the sonication experiments. Simulations yield low-velocity values for the Young's modulus of 6.0 GPa. Without protein optimization, these mechanical parameters are similar to those of spider silk and Kevlar, but in contrast to spider silk, these proteins have a precisely known sequence-structure relationship. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Channel crossing: how are proteins shipped across the bacterial plasma membrane?

PubMed

Collinson, Ian; Corey, Robin A; Allen, William J

2015-10-05

The structure of the first protein-conducting channel was determined more than a decade ago. Today, we are still puzzled by the outstanding problem of protein translocation--the dynamic mechanism underlying the consignment of proteins across and into membranes. This review is an attempt to summarize and understand the energy transducing capabilities of protein-translocating machines, with emphasis on bacterial systems: how polypeptides make headway against the lipid bilayer and how the process is coupled to the free energy associated with ATP hydrolysis and the transmembrane protein motive force. In order to explore how cargo is driven across the membrane, the known structures of the protein-translocation machines are set out against the background of the historic literature, and in the light of experiments conducted in their wake. The paper will focus on the bacterial general secretory (Sec) pathway (SecY-complex), and its eukaryotic counterpart (Sec61-complex), which ferry proteins across the membrane in an unfolded state, as well as the unrelated Tat system that assembles bespoke channels for the export of folded proteins. © 2015 The Authors.

Reparametrization of Protein Force Field Nonbonded Interactions Guided by Osmotic Coefficient Measurements from Molecular Dynamics Simulations.

PubMed

Miller, Mark S; Lay, Wesley K; Li, Shuxiang; Hacker, William C; An, Jiadi; Ren, Jianlan; Elcock, Adrian H

2017-04-11

There is a small, but growing, body of literature describing the use of osmotic coefficient measurements to validate and reparametrize simulation force fields. Here we have investigated the ability of five very commonly used force field and water model combinations to reproduce the osmotic coefficients of seven neutral amino acids and five small molecules. The force fields tested include AMBER ff99SB-ILDN, CHARMM36, GROMOS54a7, and OPLS-AA, with the first of these tested in conjunction with the TIP3P and TIP4P-Ew water models. In general, for both the amino acids and the small molecules, the tested force fields produce computed osmotic coefficients that are lower than experiment; this is indicative of excessively favorable solute-solute interactions. The sole exception to this general trend is provided by GROMOS54a7 when applied to amino acids: in this case, the computed osmotic coefficients are consistently too high. Importantly, we show that all of the force fields tested can be made to accurately reproduce the experimental osmotic coefficients of the amino acids when minor modifications-some previously reported by others and some that are new to this study-are made to the van der Waals interactions of the charged terminal groups. Special care is required, however, when simulating Proline with a number of the force fields, and a hydroxyl-group specific modification is required in order to correct Serine and Threonine when simulated with AMBER ff99SB-ILDN. Interestingly, an alternative parametrization of the van der Waals interactions in the latter force field, proposed by the Nerenberg and Head-Gordon groups, is shown to immediately produce osmotic coefficients that are in excellent agreement with experiment. Overall, this study reinforces the idea that osmotic coefficient measurements can be used to identify general shortcomings in commonly used force fields' descriptions of solute-solute interactions and further demonstrates that modifications to van der Waals parameters provide a simple route to optimizing agreement with experiment.

Reparameterization of Protein Force Field Nonbonded Interactions Guided by Osmotic Coefficient Measurements from Molecular Dynamics Simulations

PubMed Central

Miller, Mark S.; Lay, Wesley K.; Li, Shuxiang; Hacker, William C.; An, Jiadi; Ren, Jianlan; Elcock, Adrian H.

2017-01-01

There is a small, but growing, body of literature describing the use of osmotic coefficient measurements to validate and reparameterize simulation force fields. Here we have investigated the ability of five very commonly used force field and water model combinations to reproduce the osmotic coefficients of seven neutral amino acids and five small molecules. The force fields tested include AMBER ff99SB-ILDN, CHARMM36, GROMOS54a7, and OPLS-AA, with the first of these tested in conjunction with the TIP3P and TIP4P-Ew water models. In general, for both the amino acids and the small molecules, the tested force fields produce computed osmotic coefficients that are lower than experiment; this is indicative of excessively favorable solute-solute interactions. The sole exception to this general trend is provided by GROMOS54a7 when applied to amino acids: in this case, the computed osmotic coefficients are consistently too high. Importantly, we show that all of the force fields tested can be made to accurately reproduce the experimental osmotic coefficients of the amino acids when minor modifications – some previously reported by others and some that are new to this study – are made to the van der Waals interactions of the charged terminal groups. Special care is required, however, when simulating Proline with a number of the force fields, and a hydroxyl-group specific modification is required in order to correct Serine and Threonine when simulated with AMBER ff99SB-ILDN. Interestingly, an alternative parameterization of the van der Waals interactions in the latter force field, proposed by the Nerenberg and Head-Gordon groups, is shown to immediately produce osmotic coefficients that are in excellent agreement with experiment. Overall, this study reinforces the idea that osmotic coefficient measurements can be used to identify general shortcomings in commonly used force fields’ descriptions of solute-solute interactions, and further demonstrates that modifications to van der Waals parameters provides a simple route to optimizing agreement with experiment. PMID:28296391

A Force-Activated Trip Switch Triggers Rapid Dissociation of a Colicin from Its Immunity Protein

PubMed Central

Farrance, Oliver E.; Hann, Eleanore; Kaminska, Renata; Housden, Nicholas G.; Derrington, Sasha R.; Kleanthous, Colin; Radford, Sheena E.; Brockwell, David J.

2013-01-01

Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (Kd≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (<20 pN) increases the rate of complex dissociation 106-fold, to a timescale (lifetime ≈10 ms) compatible with intoxication. We term this catastrophic force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21–30) whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods. PMID:23431269

Fiber networks amplify active stress

PubMed Central

Ronceray, Pierre; Broedersz, Chase P.

2016-01-01

Large-scale force generation is essential for biological functions such as cell motility, embryonic development, and muscle contraction. In these processes, forces generated at the molecular level by motor proteins are transmitted by disordered fiber networks, resulting in large-scale active stresses. Although these fiber networks are well characterized macroscopically, this stress generation by microscopic active units is not well understood. Here we theoretically study force transmission in these networks. We find that collective fiber buckling in the vicinity of a local active unit results in a rectification of stress towards strongly amplified isotropic contraction. This stress amplification is reinforced by the networks’ disordered nature, but saturates for high densities of active units. Our predictions are quantitatively consistent with experiments on reconstituted tissues and actomyosin networks and shed light on the role of the network microstructure in shaping active stresses in cells and tissue. PMID:26921325

Magnetic Susceptibility Effects and Lorentz Damping in Diamagnetic Fluids

NASA Technical Reports Server (NTRS)

Ramachandran, Narayanan; Leslie, Fred W.

2000-01-01

A great number of crystals (semi-conductor and protein) grown in space are plagued by convective motions which contribute to structural flaws. The character of these instabilities is not well understood but is associated with density variations in the presence of residual gravity and g-jitter. Both static and dynamic (rotating or travelling wave) magnetic fields can be used to reduce the effects of convection in materials processing. In semi-conductor melts, due to their relatively high electrical conductivity, the induced Lorentz force can be effectively used to curtail convective effects. In melts/solutions with reduced electrical conductivity, such as aqueous solutions used in solution crystal growth, protein crystal growth and/or model fluid experiments for simulating melt growth, however, the variation of the magnetic susceptibility with temperature and/or concentration can be utilized to better damp fluid convection than the Lorentz force method. This paper presents a comprehensive, comparative numerical study of the relative damping effects using static magnetic fields and gradients in a simple geometry subjected to a thermal gradient. The governing equations are formulated in general terms and then simplified for the numerical calculations. Operational regimes, based on the best damping technique for different melts/solutions are identified based on fluid properties. Comparisons are provided between the numerical results and available results from experiments in surveyed literature.

Magneto-capillary valve for integrated purification and enrichment of nucleic acids and proteins.

PubMed

den Dulk, Remco C; Schmidt, Kristiane A; Sabatté, Gwénola; Liébana, Susana; Prins, Menno W J

2013-01-07

We describe the magneto-capillary valve (MCV) technology, a flexible approach for integrated biological sample preparation within the concept of stationary microfluidics. Rather than moving liquids in a microfluidic device, discrete units of liquid are present at fixed positions in the device and magnetic particles are actuated between the fluids. The MCV concept is characterized by the use of two planar surfaces at a capillary mutual distance, with specific features to confine the fluids by capillary forces, and the use of a gas or a phase-change material separating the stationary aqueous liquids. We have studied the physics of magneto-capillary valving by quantifying the magnetic force as a function of time and position, which reveals the balance of magnetic, capillary and frictional forces in the system. By purification experiments with a fluorescent tracer we have measured the amount of co-transported liquid, which is a key parameter for efficient purification. To demonstrate the versatility of the technology, several MCV device architectures were tested in a series of biological assays, showing the purification and enrichment of nucleic acids and proteins. Target recovery comparable to non-miniaturized commercial kits was observed for the extraction of DNA from human cells in buffer, using a device architecture with patterned air valves. Experiments using an enrichment module and patterned air valves demonstrate a 40-fold effective enrichment of DNA in buffer. DNA was also successfully purified from blood plasma using paraffin phase-change valves. Finally, the enrichment of a protein biomarker (prostate-specific antigen) using geometrical air valves resulted in a 7-fold increase of detection signal. The MCV technology is versatile, offers extensive freedom for the design of fully integrated systems, and is expected to be manufacturable in a cost-effective way. We conclude that the MCV technology can become an important enabling technology for point-of-care systems with sample in-result out performance.

Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

NASA Astrophysics Data System (ADS)

Strauss, Joshua; Liu, Yatao; Camesano, Terri A.

2009-09-01

Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ratto, T V; Rudd, R E; Langry, K C

We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, theymore » do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.« less

A New Generation of Crystallographic Validation Tools for the Protein Data Bank

PubMed Central

Read, Randy J.; Adams, Paul D.; Arendall, W. Bryan; Brunger, Axel T.; Emsley, Paul; Joosten, Robbie P.; Kleywegt, Gerard J.; Krissinel, Eugene B.; Lütteke, Thomas; Otwinowski, Zbyszek; Perrakis, Anastassis; Richardson, Jane S.; Sheffler, William H.; Smith, Janet L.; Tickle, Ian J.; Vriend, Gert; Zwart, Peter H.

2011-01-01

Summary This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators. PMID:22000512

A new generation of crystallographic validation tools for the protein data bank.

PubMed

Read, Randy J; Adams, Paul D; Arendall, W Bryan; Brunger, Axel T; Emsley, Paul; Joosten, Robbie P; Kleywegt, Gerard J; Krissinel, Eugene B; Lütteke, Thomas; Otwinowski, Zbyszek; Perrakis, Anastassis; Richardson, Jane S; Sheffler, William H; Smith, Janet L; Tickle, Ian J; Vriend, Gert; Zwart, Peter H

2011-10-12

This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators. Copyright © 2011 Elsevier Ltd. All rights reserved.

β-sheet-like formation during the mechanical unfolding of prion protein

NASA Astrophysics Data System (ADS)

Tao, Weiwei; Yoon, Gwonchan; Cao, Penghui; Eom, Kilho; Park, Harold S.

2015-09-01

Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrPC, whose misfolded form PrPSc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.

β-sheet-like formation during the mechanical unfolding of prion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao, Weiwei; Cao, Penghui; Park, Harold S., E-mail: parkhs@bu.edu

2015-09-28

Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP{sup C}, whose misfolded form PrP{sup Sc} can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundredsmore » of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.« less

Measurements of Attractive Forces between Proteins and End-Grafted Poly(Ethylene Glycol) Chains

NASA Astrophysics Data System (ADS)

Sheth, S. R.; Leckband, D.

1997-08-01

The surface force apparatus was used to measure directly the molecular forces between streptavidin and lipid bilayers displaying grafted Mr 2,000 poly(ethylene glycol) (PEG). These measurements provide direct evidence for the formation of relatively strong attractive forces between PEG and protein. At low compressive loads, the forces were repulsive, but they became attractive when the proteins were pressed into the polymer layer at higher loads. The adhesion was sufficiently robust that separation of the streptavidin and PEG uprooted anchored polymer from the supporting membrane. These interactions altered the properties of the grafted chains. After the onset of the attraction, the polymer continued to bind protein for several hours. The changes were not due to protein denaturation. These data demonstrate directly that the biological activity of PEG is not due solely to properties of simple polymers such as the excluded volume. It is also coupled to the competitive interactions between solvent and other materials such as proteins for the chain segments and to the ability of this material to adopt higher order intrachain structures.

A Force Balanced Fragmentation Method for ab Initio Molecular Dynamic Simulation of Protein.

PubMed

Xu, Mingyuan; Zhu, Tong; Zhang, John Z H

2018-01-01

A force balanced generalized molecular fractionation with conjugate caps (FB-GMFCC) method is proposed for ab initio molecular dynamic simulation of proteins. In this approach, the energy of the protein is computed by a linear combination of the QM energies of individual residues and molecular fragments that account for the two-body interaction of hydrogen bond between backbone peptides. The atomic forces on the caped H atoms were corrected to conserve the total force of the protein. Using this approach, ab initio molecular dynamic simulation of an Ace-(ALA) 9 -NME linear peptide showed the conservation of the total energy of the system throughout the simulation. Further a more robust 110 ps ab initio molecular dynamic simulation was performed for a protein with 56 residues and 862 atoms in explicit water. Compared with the classical force field, the ab initio molecular dynamic simulations gave better description of the geometry of peptide bonds. Although further development is still needed, the current approach is highly efficient, trivially parallel, and can be applied to ab initio molecular dynamic simulation study of large proteins.

Protein adsorption onto CF(3)-terminated oligo(ethylene glycol) containing self-assembled monolayers (SAMs): the influence of ionic strength and electrostatic forces.

PubMed

Bonnet, Nelly; O'Hagan, David; Hähner, Georg

2010-05-07

Oligo(ethylene glycol) (OEG) containing self-assembled monolayers (SAMs) on gold are known for their protein resistant properties. The underlying molecular mechanisms and the contributions of the interactions involved, however, are still not completely understood. It is known that electrostatic, van der Waals, hydrophobic, and hydration forces all play a role in the interaction between proteins and surfaces, but it is difficult to study their influence separately and to quantify their contributions. In the present study we investigate five different OEG containing SAMs and the influence of the ionic strength and the electrostatic component on the amount of a negatively charged protein (fibrinogen) that adsorbs onto them. Atomic force microscopy (AFM) was employed to record force-distance curves with hydrophobic probes depending on the ion concentration, and the amount of the protein that adsorbs relative to a hydrophobic surface was quantified using ellipsometry. The findings suggest that electrostatic forces can create a very low energy barrier thus only slightly decreasing the number of negatively charged proteins in solution with sufficient energy to approach the surface closely, and have a rather small influence on the amount that adsorbs. The films we investigated were not protein resistant. This supports other studies, reporting that a strong short-range repulsion as for example caused by hydration forces is required to make these films resistant to the non-specific adsorption of proteins.

Protein denaturants at aqueous-hydrophobic interfaces: self-consistent correlation between induced interfacial fluctuations and denaturant stability at the interface.

PubMed

Cui, Di; Ou, Shu-Ching; Patel, Sandeep

2015-01-08

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous-hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm(+)) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein-water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid-vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous-hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A 2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss 2013, 160, 89).

Quantifying protein microstructure and electrostatic effects on the change in Gibbs free energy of binding in immobilized metal affinity chromatography.

PubMed

Pathange, Lakshmi P; Bevan, David R; Zhang, Chenming

2008-03-01

Electrostatic forces play a major role in maintaining both structural and functional properties of proteins. A major component of protein electrostatics is the interactions between the charged or titratable amino acid residues (e.g., Glu, Lys, and His), whose pK(a) (or the change of the pK(a)) value could be used to study protein electrostatics. Here, we report the study of electrostatic forces through experiments using a well-controlled model protein (T4 lysozyme) and its variants. We generated 10 T4 lysozyme variants, in which the electrostatic environment of the histidine residue was perturbed by altering charged and neutral amino acid residues at various distances from the histidine (probe) residue. The electrostatic perturbations were theoretically quantified by calculating the change in free energy (DeltaDeltaG(E)) using Coulomb's law. On the other hand, immobilized metal affinity chromatography (IMAC) was used to quantify these perturbations in terms of protein binding strength or change in free energy of binding (DeltaDeltaG(B)), which varies from -0.53 to 0.99 kcal/mol. For most of the variants, there is a good correlation (R(2) = 0.97) between the theoretical DeltaDeltaG(E) and experimental DeltaDeltaG(B) values. However, there are three deviant variants, whose histidine residue was found to be involved in site-specific interactions (e.g., ion pair and steric hindrance), which were further investigated by molecular dynamics simulation. This report demonstrates that the electrostatic (DeltaDeltaG(Elec)) and microstructural effects (DeltaDeltaG(Micro)) in a protein can be quantified by IMAC through surface histidine mediated protein-metal ion interaction and that the unique microstructure around a histidine residue can be identified by identifying the abnormal binding behaviors during IMAC.

[Characteristics of tenocyte adhesion to biologically-modified surface of polymer].

PubMed

Qin, Tingwu; Yang, Zhiming; Xie, Huiqi; Li, Hong; Qin, Jian; Wu, Zezhi; Xu, Shirong; Cai, Shaoxi

2002-12-01

In this study we examined the in vitro characteristics of tenocyte adhesion to biologically-modified surface of polymer. Polylactic-co-glycolic acid (PLGA) 85/15 films were prepared by a solvent-casting technique. Each film was adhered onto the bottom of a chamber. The film was precoated with poly-D-lysine (PDL), and then coated with serum-free F12 medium containing various concentrations of fibronectin (FN), type I collagen (CN I), and insulin-like growth factor1 (IGF-1). The monoclonal antibodies (to FN and to CN I) with various dilutions were used to inhibit attachment of tenocytes to surface precoated with FN or CN I. Human embryonic tendon cells (HETCs) and transformed human embryonic tendon cells (THETCs) were used as the seeding cells. The system used for the measurement of adhesion force was the micropipette aspiration experiment system. The micropipette was manipulated to aspirate a small portion of the tenocyte body by using a small aspiration pressure. Then the pipette was pulled away from the adhesion area by micromanipulation. The minimum force required to detach the tenocyte from the substrate was defined as the adhesion force. The results showed that modification of FN or CN I by precoating significantly enhanced attachment of tenocytes to surface of polymer (P < 0.05). As antibodies to FN or CN I were added to a polymer film precoated with FN or CN I, the adhesion force decreased significantly (P < 0.05). We concluded that the specific adhesion forces of tenocytes to extracellular matrix adhesion proteins (FN and CN I) had coordinated action and showed good dependence on their precoating concentrations, and were inhibited by the antibodies to these adhesion proteins. Films precoated with IGF-1 strongly accelerated the adhesion of tenocytes to polymer. These results indicate that the specific adhesion of tenocytes to polymer can be promoted by coating extracellular matrix adhesive proteins and insulin-like growth factor1. It is of great importance to construct tissue-engineered tendon.

A force-based protein biochip

NASA Astrophysics Data System (ADS)

Blank, K.; Mai, T.; Gilbert, I.; Schiffmann, S.; Rankl, J.; Zivin, R.; Tackney, C.; Nicolaus, T.; Spinnler, K.; Oesterhelt, F.; Benoit, M.; Clausen-Schaumann, H.; Gaub, H. E.

2003-09-01

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Protein-Ligand Interaction Detection with a Novel Method of Transient Induced Molecular Electronic Spectroscopy (TIMES): Experimental and Theoretical Studies.

PubMed

Zhang, Tiantian; Wei, Tao; Han, Yuanyuan; Ma, Heng; Samieegohar, Mohammadreza; Chen, Ping-Wei; Lian, Ian; Lo, Yu-Hwa

2016-11-23

Protein-ligand interaction detection without disturbances (e.g., surface immobilization, fluorescent labeling, and crystallization) presents a key question in protein chemistry and drug discovery. The emergent technology of transient induced molecular electronic spectroscopy (TIMES), which incorporates a unique design of microfluidic platform and integrated sensing electrodes, is designed to operate in a label-free and immobilization-free manner to provide crucial information for protein-ligand interactions in relevant physiological conditions. Through experiments and theoretical simulations, we demonstrate that the TIMES technique actually detects protein-ligand binding through signals generated by surface electric polarization. The accuracy and sensitivity of experiments were demonstrated by precise measurements of dissociation constant of lysozyme and N -acetyl-d-glucosamine (NAG) ligand and its trimer, NAG 3 . Computational fluid dynamics (CFD) computation is performed to demonstrate that the surface's electric polarization signal originates from the induced image charges during the transition state of surface mass transport, which is governed by the overall effects of protein concentration, hydraulic forces, and surface fouling due to protein adsorption. Hybrid atomistic molecular dynamics (MD) simulations and free energy computation show that ligand binding affects lysozyme structure and stability, producing different adsorption orientation and surface polarization to give the characteristic TIMES signals. Although the current work is focused on protein-ligand interactions, the TIMES method is a general technique that can be applied to study signals from reactions between many kinds of molecules.

Mechanical response and buckling of a polymer simulation model of the cell nucleus

NASA Astrophysics Data System (ADS)

Banigan, Edward; Stephens, Andrew; Marko, John

The cell nucleus must robustly resist extra- and intracellular forces to maintain genome architecture. Micromanipulation experiments measuring nuclear mechanical response reveal that the nucleus has two force response regimes: a linear short-extension response due to the chromatin interior and a stiffer long-extension response from lamin A, comprising the intermediate filament protein shell. To explain these results, we developed a quantitative simulation model with realistic parameters for chromatin and the lamina. Our model predicts that crosslinking between chromatin and the lamina is essential for responding to small strains and that changes to the interior topological organization can alter the mechanical response of the whole nucleus. Thus, chromatin polymer elasticity, not osmotic pressure, is the dominant regulator of this force response. Our model reveals a novel buckling transition for polymer shells: as force increases, the shell buckles transverse to the applied force. This transition, which arises from topological constrains in the lamina, can be mitigated by tuning the properties of the chromatin interior. Thus, we find that the genome is a resistive mechanical element that can be tuned by its organization and connectivity to the lamina.

Slow-Down in Diffusion in Crowded Protein Solutions Correlates with Transient Cluster Formation.

PubMed

Nawrocki, Grzegorz; Wang, Po-Hung; Yu, Isseki; Sugita, Yuji; Feig, Michael

2017-12-14

For a long time, the effect of a crowded cellular environment on protein dynamics has been largely ignored. Recent experiments indicate that proteins diffuse more slowly in a living cell than in a diluted solution, and further studies suggest that the diffusion depends on the local surroundings. Here, detailed insight into how diffusion depends on protein-protein contacts is presented based on extensive all-atom molecular dynamics simulations of concentrated villin headpiece solutions. After force field adjustments in the form of increased protein-water interactions to reproduce experimental data, translational and rotational diffusion was analyzed in detail. Although internal protein dynamics remained largely unaltered, rotational diffusion was found to slow down more significantly than translational diffusion as the protein concentration increased. The decrease in diffusion is interpreted in terms of a transient formation of protein clusters. These clusters persist on sub-microsecond time scales and follow distributions that increasingly shift toward larger cluster size with increasing protein concentrations. Weighting diffusion coefficients estimated for different clusters extracted from the simulations with the distribution of clusters largely reproduces the overall observed diffusion rates, suggesting that transient cluster formation is a primary cause for a slow-down in diffusion upon crowding with other proteins.

Systematic Validation of Protein Force Fields against Experimental Data

PubMed Central

Eastwood, Michael P.; Dror, Ron O.; Shaw, David E.

2012-01-01

Molecular dynamics simulations provide a vehicle for capturing the structures, motions, and interactions of biological macromolecules in full atomic detail. The accuracy of such simulations, however, is critically dependent on the force field—the mathematical model used to approximate the atomic-level forces acting on the simulated molecular system. Here we present a systematic and extensive evaluation of eight different protein force fields based on comparisons of experimental data with molecular dynamics simulations that reach a previously inaccessible timescale. First, through extensive comparisons with experimental NMR data, we examined the force fields' abilities to describe the structure and fluctuations of folded proteins. Second, we quantified potential biases towards different secondary structure types by comparing experimental and simulation data for small peptides that preferentially populate either helical or sheet-like structures. Third, we tested the force fields' abilities to fold two small proteins—one α-helical, the other with β-sheet structure. The results suggest that force fields have improved over time, and that the most recent versions, while not perfect, provide an accurate description of many structural and dynamical properties of proteins. PMID:22384157

Exchange of adsorbed serum proteins during adhesion of Staphylococcus aureus to an abiotic surface and Candida albicans hyphae--an AFM study.

PubMed

Ovchinnikova, Ekaterina S; van der Mei, Henny C; Krom, Bastiaan P; Busscher, Henk J

2013-10-01

Staphylococcus aureus and Candida albicans are the second and third most commonly isolated microorganisms in hospital-related-infections, that are often multi-species in nature causing high morbidity and mortality. Here, adhesion forces between a S. aureus strain and abiotic (tissue-culture-polystyrene, TCPS) or partly biotic (TCPS with adhering hyphae of C. albicans) surfaces were investigated in presence of fetal-bovine-serum or individual serum proteins and related with staphylococcal adhesion. Atomic-force-microscopy was used to measure adhesion forces between S. aureus and the abiotic and biotic surfaces. Adsorption of individual serum proteins like albumin and apo-transferrin to abiotic TCPS surfaces during 60min, impeded development of strong adhesion forces as compared to fibronectin, while 60min adsorption of proteins from fetal-bovine-serum yielded a decrease in adhesion force from -5.7nN in phosphate-buffered-saline to -0.6nN. Adsorption of albumin and apo-transferrin also decreased staphylococcal adhesion forces to hyphae as compared with fibronectin. During 60min exposure to fetal-bovine-serum however, initial (5min protein adsorption) staphylococcal adhesion forces were low (-1.6nN), but strong adhesion forces of around -5.5nN were restored within 60min. This suggests for the first time that in whole fetal-bovine-serum exchange of non-adhesive proteins by fibronectin occurs on biotic C. albicans hyphal surfaces. No evidence was found for such protein exchange on abiotic TCPS surfaces. Staphylococcal adhesion of abiotic and biotic surfaces varied in line with the adhesion forces and was low on TCPS in presence of fetal-bovine-serum. On partly biotic TCPS, staphylococci aggregated in presence of fetal-bovine-serum around adhering C. albicans hyphae. Copyright © 2013 Elsevier B.V. All rights reserved.

Unconstrained Structure Formation in Coarse-Grained Protein Simulations

NASA Astrophysics Data System (ADS)

Bereau, Tristan

The ability of proteins to fold into well-defined structures forms the basis of a wide variety of biochemical functions in and out of the cell membrane. Many of these processes, however, operate at time- and length-scales that are currently unattainable by all-atom computer simulations. To cope with this difficulty, increasingly more accurate and sophisticated coarse-grained models are currently being developed. In the present thesis, we introduce a solvent-free coarse-grained model for proteins. Proteins are modeled by four beads per amino acid, providing enough backbone resolution to allow for accurate sampling of local conformations. It relies on simple interactions that emphasize structure, such as hydrogen bonds and hydrophobicity. Realistic alpha/beta content is achieved by including an effective nearest-neighbor dipolar interaction. Parameters are tuned to reproduce both local conformations and tertiary structures. By studying both helical and extended conformations we make sure the force field is not biased towards any particular secondary structure. Without any further adjustments or bias a realistic oligopeptide aggregation scenario is observed. The model is subsequently applied to various biophysical problems: (i) kinetics of folding of two model peptides, (ii) large-scale amyloid-beta oligomerization, and (iii) protein folding cooperativity. The last topic---defined by the nature of the finite-size thermodynamic transition exhibited upon folding---was investigated from a microcanonical perspective: the accurate evaluation of the density of states can unambiguously characterize the nature of the transition, unlike its corresponding canonical analysis. Extending the results of lattice simulations and theoretical models, we find that it is the interplay between secondary structure and the loss of non-native tertiary contacts which determines the nature of the transition. Finally, we combine the peptide model with a high-resolution, solvent-free, lipid model. The lipid force field was systematically tuned to reproduce the structural and mechanical properties of phosphatidylcholine bilayers. The two models were cross-parametrized against atomistic potential of mean force curves for the insertion of single amino acid side chains into a bilayer. Coarse-grained transmembrane protein simulations were then compared with experiments and atomistic simulations to validate the force field. The transferability of the two models across amino acid sequences and lipid species permits the investigation of a wide variety of scenarios, while the absence of explicit solvent allows for studies of large-scale phenomena.

Covalent Immobilization of Microtubules on Glass Surfaces for Molecular Motor Force Measurements and Other Single-Molecule Assays

PubMed Central

Nicholas, Matthew P.; Rao, Lu; Gennerich, Arne

2014-01-01

Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety of microscopy experiments in which MTs are used as substrates for MT-associated proteins, such as the molecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fluorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers experiments with MT-associated molecular motors and may also be useful in other assays requiring surface-immobilized proteins. PMID:24633798

How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory.

PubMed

Mondal, Jagannath; Halverson, Duncan; Li, Isaac T S; Stirnemann, Guillaume; Walker, Gilbert C; Berne, Bruce J

2015-07-28

It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford-Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard-Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system.

How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory

PubMed Central

Mondal, Jagannath; Halverson, Duncan; Li, Isaac T. S.; Stirnemann, Guillaume; Walker, Gilbert C.; Berne, Bruce J.

2015-01-01

It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford–Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard–Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system. PMID:26170324

Forces in yeast flocculation

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

2015-01-01

In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

Order statistics inference for describing topological coupling and mechanical symmetry breaking in multidomain proteins

NASA Astrophysics Data System (ADS)

Kononova, Olga; Jones, Lee; Barsegov, V.

2013-09-01

Cooperativity is a hallmark of proteins, many of which show a modular architecture comprising discrete structural domains. Detecting and describing dynamic couplings between structural regions is difficult in view of the many-body nature of protein-protein interactions. By utilizing the GPU-based computational acceleration, we carried out simulations of the protein forced unfolding for the dimer WW - WW of the all-β-sheet WW domains used as a model multidomain protein. We found that while the physically non-interacting identical protein domains (WW) show nearly symmetric mechanical properties at low tension, reflected, e.g., in the similarity of their distributions of unfolding times, these properties become distinctly different when tension is increased. Moreover, the uncorrelated unfolding transitions at a low pulling force become increasingly more correlated (dependent) at higher forces. Hence, the applied force not only breaks "the mechanical symmetry" but also couples the physically non-interacting protein domains forming a multi-domain protein. We call this effect "the topological coupling." We developed a new theory, inspired by order statistics, to characterize protein-protein interactions in multi-domain proteins. The method utilizes the squared-Gaussian model, but it can also be used in conjunction with other parametric models for the distribution of unfolding times. The formalism can be taken to the single-molecule experimental lab to probe mechanical cooperativity and domain communication in multi-domain proteins.

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH.

PubMed

Tian, Ye; Schwieters, Charles D; Opella, Stanley J; Marassi, Francesca M

2017-01-01

Structure determination of proteins by NMR is unique in its ability to measure restraints, very accurately, in environments and under conditions that closely mimic those encountered in vivo. For example, advances in solid-state NMR methods enable structure determination of membrane proteins in detergent-free lipid bilayers, and of large soluble proteins prepared by sedimentation, while parallel advances in solution NMR methods and optimization of detergent-free lipid nanodiscs are rapidly pushing the envelope of the size limit for both soluble and membrane proteins. These experimental advantages, however, are partially squandered during structure calculation, because the commonly used force fields are purely repulsive and neglect solvation, Van der Waals forces and electrostatic energy. Here we describe a new force field, and updated energy functions, for protein structure calculations with EEFx implicit solvation, electrostatics, and Van der Waals Lennard-Jones forces, in the widely used program Xplor-NIH. The new force field is based primarily on CHARMM22, facilitating calculations with a wider range of biomolecules. The new EEFx energy function has been rewritten to enable OpenMP parallelism, and optimized to enhance computation efficiency. It implements solvation, electrostatics, and Van der Waals energy terms together, thus ensuring more consistent and efficient computation of the complete nonbonded energy lists. Updates in the related python module allow detailed analysis of the interaction energies and associated parameters. The new force field and energy function work with both soluble proteins and membrane proteins, including those with cofactors or engineered tags, and are very effective in situations where there are sparse experimental restraints. Results obtained for NMR-restrained calculations with a set of five soluble proteins and five membrane proteins show that structures calculated with EEFx have significant improvements in accuracy, precision, and conformation, and that structure refinement can be obtained by short relaxation with EEFx to obtain improvements in these key metrics. These developments broaden the range of biomolecular structures that can be calculated with high fidelity from NMR restraints.

Combining experimental and simulation data of molecular processes via augmented Markov models.

PubMed

Olsson, Simon; Wu, Hao; Paul, Fabian; Clementi, Cecilia; Noé, Frank

2017-08-01

Accurate mechanistic description of structural changes in biomolecules is an increasingly important topic in structural and chemical biology. Markov models have emerged as a powerful way to approximate the molecular kinetics of large biomolecules while keeping full structural resolution in a divide-and-conquer fashion. However, the accuracy of these models is limited by that of the force fields used to generate the underlying molecular dynamics (MD) simulation data. Whereas the quality of classical MD force fields has improved significantly in recent years, remaining errors in the Boltzmann weights are still on the order of a few [Formula: see text], which may lead to significant discrepancies when comparing to experimentally measured rates or state populations. Here we take the view that simulations using a sufficiently good force-field sample conformations that are valid but have inaccurate weights, yet these weights may be made accurate by incorporating experimental data a posteriori. To do so, we propose augmented Markov models (AMMs), an approach that combines concepts from probability theory and information theory to consistently treat systematic force-field error and statistical errors in simulation and experiment. Our results demonstrate that AMMs can reconcile conflicting results for protein mechanisms obtained by different force fields and correct for a wide range of stationary and dynamical observables even when only equilibrium measurements are incorporated into the estimation process. This approach constitutes a unique avenue to combine experiment and computation into integrative models of biomolecular structure and dynamics.

Compressive Force Spectroscopy: From Living Cells to Single Proteins.

PubMed

Wang, Jiabin; Liu, Meijun; Shen, Yi; Sun, Jielin; Shao, Zhifeng; Czajkowsky, Daniel Mark

2018-03-23

One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.

Protein Adsorption and Deposition onto Microfiltration Membranes: The Role of Solute-Solid Interactions.

PubMed

Martínez; Martín; Prádanos; Calvo; Palacio; Hernández

2000-01-15

The mass of gamma-globulin fouling an Anodisc alumina membrane with a nominal pore diameter of 0.1 µm has been measured at several concentrations and pHs. This fouling resulted from filtering through the membrane in a continuous recirculation device. The low-concentration fouling can be attributed mainly to adsorption. The complete concentration dependence of fouling mass has been obtained and fitted to a Freundlich heterogeneous isotherm, from which the pH dependence of active fouling sites and energies has been also obtained. Adsorption is studied as a function of the electrostatic forces between the solute and the membrane. A sharp maximum in the adsorbed mass for zero electrostatic force is observed. At high concentrations, accumulation plays a relevant role at alkaline pH, as confirmed by flux decay experiments, retention measurements, and AFM (atomic force microscopy) pictures. Copyright 2000 Academic Press.

TMFF-A Two-Bead Multipole Force Field for Coarse-Grained Molecular Dynamics Simulation of Protein.

PubMed

Li, Min; Liu, Fengjiao; Zhang, John Z H

2016-12-13

Coarse-grained (CG) models are desirable for studying large and complex biological systems. In this paper, we propose a new two-bead multipole force field (TMFF) in which electric multipoles up to the quadrupole are included in the CG force field. The inclusion of electric multipoles in the proposed CG force field enables a more realistic description of the anisotropic electrostatic interactions in the protein system and, thus, provides an improvement over the standard isotropic two-bead CG models. In order to test the accuracy of the new CG force field model, extensive molecular dynamics simulations were carried out for a series of benchmark protein systems. These simulation studies showed that the TMFF model can realistically reproduce the structural and dynamical properties of proteins, as demonstrated by the close agreement of the CG results with those from the corresponding all-atom simulations in terms of root-mean-square deviations (RMSDs) and root-mean-square fluctuations (RMSFs) of the protein backbones. The current two-bead model is highly coarse-grained and is 50-fold more efficient than all-atom method in MD simulation of proteins in explicit water.

Tunneling Kinetics and Nonadiabatic Proton-Coupled Electron Transfer in Proteins: The Effect of Electric Fields and Anharmonic Donor-Acceptor Interactions.

PubMed

Salna, Bridget; Benabbas, Abdelkrim; Russo, Douglas; Champion, Paul M

2017-07-20

A proper description of proton donor-acceptor (D-A) distance fluctuations is crucial for understanding tunneling in proton-coupled electron transport (PCET). The typical harmonic approximation for the D-A potential results in a Gaussian probability distribution, which does not appropriately reflect the electronic repulsion forces that increase the energetic cost of sampling shorter D-A distances. Because these shorter distances are the primary channel for thermally activated tunneling, the analysis of tunneling kinetics depends sensitively on the inherently anharmonic nature of the D-A interaction. Thus, we have used quantum chemical calculations to account for the D-A interaction and developed an improved model for the analysis of experimental tunneling kinetics. Strong internal electric fields are also considered and found to contribute significantly to the compressive forces when the D-A distance distribution is positioned below the van der Waals contact distance. This model is applied to recent experiments on the wild type (WT) and a double mutant (DM) of soybean lipoxygenase-1 (SLO). The compressive force necessary to prepare the tunneling-active distribution in WT SLO is found to fall in the ∼ nN range, which greatly exceeds the measured values of molecular motor and protein unfolding forces. This indicates that ∼60-100 MV/cm electric fields, aligned along the D-A bond axis, must be generated by an enzyme conformational interconversion that facilitates the PCET tunneling reaction. Based on the absolute value of the measured tunneling rate, and using previously calculated values of the electronic matrix element, the population of this tunneling-active conformation is found to lie in the range 10 -5 -10 -7 , indicating this is a rare structural fluctuation that falls well below the detection threshold of recent ENDOR experiments. Additional analysis of the DM tunneling kinetics leads to a proposal that a disordered (high entropy) conformation could be tunneling-active due to its broad range of sampled D-A distances.

How to understand atomistic molecular dynamics simulations of RNA and protein-RNA complexes?

PubMed

Šponer, Jiří; Krepl, Miroslav; Banáš, Pavel; Kührová, Petra; Zgarbová, Marie; Jurečka, Petr; Havrila, Marek; Otyepka, Michal

2017-05-01

We provide a critical assessment of explicit-solvent atomistic molecular dynamics (MD) simulations of RNA and protein/RNA complexes, written primarily for non-specialists with an emphasis to explain the limitations of MD. MD simulations can be likened to hypothetical single-molecule experiments starting from single atomistic conformations and investigating genuine thermal sampling of the biomolecules. The main advantage of MD is the unlimited temporal and spatial resolution of positions of all atoms in the simulated systems. Fundamental limitations are the short physical time-scale of simulations, which can be partially alleviated by enhanced-sampling techniques, and the highly approximate atomistic force fields describing the simulated molecules. The applicability and present limitations of MD are demonstrated on studies of tetranucleotides, tetraloops, ribozymes, riboswitches and protein/RNA complexes. Wisely applied simulations respecting the approximations of the model can successfully complement structural and biochemical experiments. WIREs RNA 2017, 8:e1405. doi: 10.1002/wrna.1405 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

Protein Denaturants at Aqueous–Hydrophobic Interfaces: Self-Consistent Correlation between Induced Interfacial Fluctuations and Denaturant Stability at the Interface

PubMed Central

2015-01-01

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous–hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm+) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein–water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid–vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous–hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss2013, 160, 89). PMID:25536388

MTS-MD of Biomolecules Steered with 3D-RISM-KH Mean Solvation Forces Accelerated with Generalized Solvation Force Extrapolation.

PubMed

Omelyan, Igor; Kovalenko, Andriy

2015-04-14

We developed a generalized solvation force extrapolation (GSFE) approach to speed up multiple time step molecular dynamics (MTS-MD) of biomolecules steered with mean solvation forces obtained from the 3D-RISM-KH molecular theory of solvation (three-dimensional reference interaction site model with the Kovalenko-Hirata closure). GSFE is based on a set of techniques including the non-Eckart-like transformation of coordinate space separately for each solute atom, extension of the force-coordinate pair basis set followed by selection of the best subset, balancing the normal equations by modified least-squares minimization of deviations, and incremental increase of outer time step in motion integration. Mean solvation forces acting on the biomolecule atoms in conformations at successive inner time steps are extrapolated using a relatively small number of best (closest) solute atomic coordinates and corresponding mean solvation forces obtained at previous outer time steps by converging the 3D-RISM-KH integral equations. The MTS-MD evolution steered with GSFE of 3D-RISM-KH mean solvation forces is efficiently stabilized with our optimized isokinetic Nosé-Hoover chain (OIN) thermostat. We validated the hybrid MTS-MD/OIN/GSFE/3D-RISM-KH integrator on solvated organic and biomolecules of different stiffness and complexity: asphaltene dimer in toluene solvent, hydrated alanine dipeptide, miniprotein 1L2Y, and protein G. The GSFE accuracy and the OIN efficiency allowed us to enlarge outer time steps up to huge values of 1-4 ps while accurately reproducing conformational properties. Quasidynamics steered with 3D-RISM-KH mean solvation forces achieves time scale compression of conformational changes coupled with solvent exchange, resulting in further significant acceleration of protein conformational sampling with respect to real time dynamics. Overall, this provided a 50- to 1000-fold effective speedup of conformational sampling for these systems, compared to conventional MD with explicit solvent. We have been able to fold the miniprotein from a fully denatured, extended state in about 60 ns of quasidynamics steered with 3D-RISM-KH mean solvation forces, compared to the average physical folding time of 4-9 μs observed in experiment.

COFFDROP: A Coarse-Grained Nonbonded Force Field for Proteins Derived from All-Atom Explicit-Solvent Molecular Dynamics Simulations of Amino Acids.

PubMed

Andrews, Casey T; Elcock, Adrian H

2014-11-11

We describe the derivation of a set of bonded and nonbonded coarse-grained (CG) potential functions for use in implicit-solvent Brownian dynamics (BD) simulations of proteins derived from all-atom explicit-solvent molecular dynamics (MD) simulations of amino acids. Bonded potential functions were derived from 1 μs MD simulations of each of the 20 canonical amino acids, with histidine modeled in both its protonated and neutral forms; nonbonded potential functions were derived from 1 μs MD simulations of every possible pairing of the amino acids (231 different systems). The angle and dihedral probability distributions and radial distribution functions sampled during MD were used to optimize a set of CG potential functions through use of the iterative Boltzmann inversion (IBI) method. The optimized set of potential functions-which we term COFFDROP (COarse-grained Force Field for Dynamic Representation Of Proteins)-quantitatively reproduced all of the "target" MD distributions. In a first test of the force field, it was used to predict the clustering behavior of concentrated amino acid solutions; the predictions were directly compared with the results of corresponding all-atom explicit-solvent MD simulations and found to be in excellent agreement. In a second test, BD simulations of the small protein villin headpiece were carried out at concentrations that have recently been studied in all-atom explicit-solvent MD simulations by Petrov and Zagrovic ( PLoS Comput. Biol. 2014 , 5 , e1003638). The anomalously strong intermolecular interactions seen in the MD study were reproduced in the COFFDROP simulations; a simple scaling of COFFDROP's nonbonded parameters, however, produced results in better accordance with experiment. Overall, our results suggest that potential functions derived from simulations of pairwise amino acid interactions might be of quite broad applicability, with COFFDROP likely to be especially useful for modeling unfolded or intrinsically disordered proteins.

COFFDROP: A Coarse-Grained Nonbonded Force Field for Proteins Derived from All-Atom Explicit-Solvent Molecular Dynamics Simulations of Amino Acids

PubMed Central

2015-01-01

We describe the derivation of a set of bonded and nonbonded coarse-grained (CG) potential functions for use in implicit-solvent Brownian dynamics (BD) simulations of proteins derived from all-atom explicit-solvent molecular dynamics (MD) simulations of amino acids. Bonded potential functions were derived from 1 μs MD simulations of each of the 20 canonical amino acids, with histidine modeled in both its protonated and neutral forms; nonbonded potential functions were derived from 1 μs MD simulations of every possible pairing of the amino acids (231 different systems). The angle and dihedral probability distributions and radial distribution functions sampled during MD were used to optimize a set of CG potential functions through use of the iterative Boltzmann inversion (IBI) method. The optimized set of potential functions—which we term COFFDROP (COarse-grained Force Field for Dynamic Representation Of Proteins)—quantitatively reproduced all of the “target” MD distributions. In a first test of the force field, it was used to predict the clustering behavior of concentrated amino acid solutions; the predictions were directly compared with the results of corresponding all-atom explicit-solvent MD simulations and found to be in excellent agreement. In a second test, BD simulations of the small protein villin headpiece were carried out at concentrations that have recently been studied in all-atom explicit-solvent MD simulations by Petrov and Zagrovic (PLoS Comput. Biol.2014, 5, e1003638). The anomalously strong intermolecular interactions seen in the MD study were reproduced in the COFFDROP simulations; a simple scaling of COFFDROP’s nonbonded parameters, however, produced results in better accordance with experiment. Overall, our results suggest that potential functions derived from simulations of pairwise amino acid interactions might be of quite broad applicability, with COFFDROP likely to be especially useful for modeling unfolded or intrinsically disordered proteins. PMID:25400526

Free energy surface of an intrinsically disordered protein: comparison between temperature replica exchange molecular dynamics and bias-exchange metadynamics.

PubMed

Zerze, Gül H; Miller, Cayla M; Granata, Daniele; Mittal, Jeetain

2015-06-09

Intrinsically disordered proteins (IDPs), which are expected to be largely unstructured under physiological conditions, make up a large fraction of eukaryotic proteins. Molecular dynamics simulations have been utilized to probe structural characteristics of these proteins, which are not always easily accessible to experiments. However, exploration of the conformational space by brute force molecular dynamics simulations is often limited by short time scales. Present literature provides a number of enhanced sampling methods to explore protein conformational space in molecular simulations more efficiently. In this work, we present a comparison of two enhanced sampling methods: temperature replica exchange molecular dynamics and bias exchange metadynamics. By investigating both the free energy landscape as a function of pertinent order parameters and the per-residue secondary structures of an IDP, namely, human islet amyloid polypeptide, we found that the two methods yield similar results as expected. We also highlight the practical difference between the two methods by describing the path that we followed to obtain both sets of data.

The molten globule state is unusually deformable under mechanical force

PubMed Central

Elms, Phillip J.; Chodera, John D.; Bustamante, Carlos; Marqusee, Susan

2012-01-01

Recently, the role of force in cellular processes has become more evident, and now with advances in force spectroscopy, the response of proteins to force can be directly studied. Such studies have found that native proteins are brittle, and thus not very deformable. Here, we examine the mechanical properties of a class of intermediates referred to as the molten globule state. Using optical trap force spectroscopy, we investigated the response to force of the native and molten globule states of apomyoglobin along different pulling axes. Unlike natively folded proteins, the molten globule state of apomyoglobin is compliant (large distance to the transition state); this large compliance means that the molten globule is more deformable and the unfolding rate is more sensitive to force (the application of force or tension will have a more dramatic effect on the unfolding rate). Our studies suggest that these are general properties of molten globules and could have important implications for mechanical processes in the cell. PMID:22355138

The mechano-sensing role of the unique SH3 insertion in plakin domains revealed by Molecular Dynamics simulations.

PubMed

Daday, Csaba; Kolšek, Katra; Gräter, Frauke

2017-09-15

The plakin family of proteins, important actors in cross-linking force-bearing structures in the cell, contain a curious SH3 domain insertion in their chain of spectrin repeats (SRs). While SH3 domains are known to mediate protein-protein interactions, here, its canonical binding site is autoinhibited by the preceding SR. Under force, however, this SH3 domain could be released, and possibly launch a signaling cascade. We performed large-scale force-probe molecular dynamics simulations, across two orders of magnitude of loading rates, to test this hypothesis, on two prominent members of the plakin family: desmoplakin and plectin, obligate proteins at desmosomes and hemidesmosomes, respectively. Our simulations show that force unravels the SRs and abolishes the autoinhibition of the SH3 domain, an event well separated from the unfolding of this domain. The SH3 domain is free and fully functional for a significant portion of the unfolding trajectories. The rupture forces required for the two proteins significantly decrease when the SH3 domain is removed, which implies that the SH3 domain also stabilizes this junction. Our results persist across all simulations, and support a force-sensing as well as a stabilizing role of the unique SH3 insertion, putting forward this protein family as a new class of mechano-sensors.

Nanoindentation of virus capsids in a molecular model

NASA Astrophysics Data System (ADS)

Cieplak, Marek; Robbins, Mark O.

2010-01-01

A molecular-level model is used to study the mechanical response of empty cowpea chlorotic mottle virus (CCMV) and cowpea mosaic virus (CPMV) capsids. The model is based on the native structure of the proteins that constitute the capsids and is described in terms of the Cα atoms. Nanoindentation by a large tip is modeled as compression between parallel plates. Plots of the compressive force versus plate separation for CCMV are qualitatively consistent with continuum models and experiments, showing an elastic region followed by an irreversible drop in force. The mechanical response of CPMV has not been studied, but the molecular model predicts an order of magnitude higher stiffness and a much shorter elastic region than for CCMV. These large changes result from small structural changes that increase the number of bonds by only 30% and would be difficult to capture in continuum models. Direct comparison of local deformations in continuum and molecular models of CCMV shows that the molecular model undergoes a gradual symmetry breaking rotation and accommodates more strain near the walls than the continuum model. The irreversible drop in force at small separations is associated with rupturing nearly all of the bonds between capsid proteins in the molecular model, while a buckling transition is observed in continuum models.

Exploration of Structural Changes in Lactose Permease on Sugar Binding and Proton Transport through Atomistic Simulations

NASA Astrophysics Data System (ADS)

Liu, Jin; Jewel, Yead; Dutta, Prashanta

2017-11-01

Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H+ symport process. Lactose/H+ symport is a highly complex process that involves large-scale protein conformational changes. The complete picture of lactose/H+ symport is largely unclear. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid. After validation, we implement the new force field to investigate the binding of a β-D-galactopyranosyl-1-thio- β-D-galactopyranoside (TDG) molecule to a wild-type LacY. Transitions from inward-facing to outward-facing conformations upon TDG binding and protonation of Glu269 have been achieved from microsecond simulations. Both the opening of the periplasmic side and closure of the cytoplasmic side of LacY are consistent with experiments. Our analysis suggest that the conformational changes of LacY are a cumulative consequence of inter-domain H-bonds breaking at the periplasmic side, inter-domain salt-bridge formation at the cytoplasmic side, as well as the TDG orientational changes during the transition. This work is supported by US National Science Foundation under Grant No. CBET-1604211.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ilieva, N., E-mail: nevena.ilieva@parallel.bas.bg; Dai, J., E-mail: daijing491@gmail.com; Sieradzan, A., E-mail: adams86@wp.pl

Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen’s dogma states that the native 3D shape of a protein is completely determined by protein’s amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolvedmore » problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix–loop–helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics.« less

Analysis of DNA interactions using single-molecule force spectroscopy.

PubMed

Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert

2013-06-01

Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

Direct folding simulation of helical proteins using an effective polarizable bond force field.

PubMed

Duan, Lili; Zhu, Tong; Ji, Changge; Zhang, Qinggang; Zhang, John Z H

2017-06-14

We report a direct folding study of seven helical proteins (, Trpcage, , C34, N36, , ) ranging from 17 to 53 amino acids through standard molecular dynamics simulations using a recently developed polarizable force field-Effective Polarizable Bond (EPB) method. The backbone RMSDs, radius of gyrations, native contacts and native helix content are in good agreement with the experimental results. Cluster analysis has also verified that these folded structures with the highest population are in good agreement with their corresponding native structures for these proteins. In addition, the free energy landscape of seven proteins in the two dimensional space comprised of RMSD and radius of gyration proved that these folded structures are indeed of the lowest energy conformations. However, when the corresponding simulations were performed using the standard (nonpolarizable) AMBER force fields, no stable folded structures were observed for these proteins. Comparison of the simulation results based on a polarizable EPB force field and a nonpolarizable AMBER force field clearly demonstrates the importance of polarization in the folding of stable helical structures.

Improved side-chain torsion potentials for the Amber ff99SB protein force field

PubMed Central

Lindorff-Larsen, Kresten; Piana, Stefano; Palmo, Kim; Maragakis, Paul; Klepeis, John L; Dror, Ron O; Shaw, David E

2010-01-01

Recent advances in hardware and software have enabled increasingly long molecular dynamics (MD) simulations of biomolecules, exposing certain limitations in the accuracy of the force fields used for such simulations and spurring efforts to refine these force fields. Recent modifications to the Amber and CHARMM protein force fields, for example, have improved the backbone torsion potentials, remedying deficiencies in earlier versions. Here, we further advance simulation accuracy by improving the amino acid side-chain torsion potentials of the Amber ff99SB force field. First, we used simulations of model alpha-helical systems to identify the four residue types whose rotamer distribution differed the most from expectations based on Protein Data Bank statistics. Second, we optimized the side-chain torsion potentials of these residues to match new, high-level quantum-mechanical calculations. Finally, we used microsecond-timescale MD simulations in explicit solvent to validate the resulting force field against a large set of experimental NMR measurements that directly probe side-chain conformations. The new force field, which we have termed Amber ff99SB-ILDN, exhibits considerably better agreement with the NMR data. Proteins 2010. © 2010 Wiley-Liss, Inc. PMID:20408171

Incorporating water-release and lateral protein interactions in modeling equilibrium adsorption for ion-exchange chromatography.

PubMed

Thrash, Marvin E; Pinto, Neville G

2006-09-08

The equilibrium adsorption of two albumin proteins on a commercial ion exchanger has been studied using a colloidal model. The model accounts for electrostatic and van der Waals forces between proteins and the ion exchanger surface, the energy of interaction between adsorbed proteins, and the contribution of entropy from water-release accompanying protein adsorption. Protein-surface interactions were calculated using methods previously reported in the literature. Lateral interactions between adsorbed proteins were experimentally measured with microcalorimetry. Water-release was estimated by applying the preferential interaction approach to chromatographic retention data. The adsorption of ovalbumin and bovine serum albumin on an anion exchanger at solution pH>pI of protein was measured. The experimental isotherms have been modeled from the linear region to saturation, and the influence of three modulating alkali chlorides on capacity has been evaluated. The heat of adsorption is endothermic for all cases studied, despite the fact that the net charge on the protein is opposite that of the adsorbing surface. Strong repulsive forces between adsorbed proteins underlie the endothermic heat of adsorption, and these forces intensify with protein loading. It was found that the driving force for adsorption is the entropy increase due to the release of water from the protein and adsorbent surfaces. It is shown that the colloidal model predicts protein adsorption capacity in both the linear and non-linear isotherm regions, and can account for the effects of modulating salt.

[Subcellular basis of disorders of the contractile capacity of the heart in L-thyroxine-induced thyrotoxicosis].

PubMed

Karsanov, N V; Melashvili, N O; Khugashvili, Z G; Mamulashvili, L D; Azrumelashvili, M I; Khaindrava, G K; Kapanadze, R V

1990-02-01

In experiments on dogs, the authors examined the functional activity of three cardiomyocyte systems responsible for contraction-relaxation (the systems of contractile proteins, calcium transport and energy supply) in the dynamics of L-thyroxine-induced toxicosis. A fall in the capacity of the contractile protein system to generate energy and to perform was shown to play the leading role in decrease of myocardial reserve forces and reduction in cardiac contractility. There was a drop in the intensity of calcium transport through the membranes of the sarcoplasmic reticulum and mitochondria and a deficiency of the direct energy source for contraction only in the late period of the disease.

Parametrization of Backbone Flexibility in a Coarse-Grained Force Field for Proteins (COFFDROP) Derived from All-Atom Explicit-Solvent Molecular Dynamics Simulations of All Possible Two-Residue Peptides.

PubMed

Frembgen-Kesner, Tamara; Andrews, Casey T; Li, Shuxiang; Ngo, Nguyet Anh; Shubert, Scott A; Jain, Aakash; Olayiwola, Oluwatoni J; Weishaar, Mitch R; Elcock, Adrian H

2015-05-12

Recently, we reported the parametrization of a set of coarse-grained (CG) nonbonded potential functions, derived from all-atom explicit-solvent molecular dynamics (MD) simulations of amino acid pairs and designed for use in (implicit-solvent) Brownian dynamics (BD) simulations of proteins; this force field was named COFFDROP (COarse-grained Force Field for Dynamic Representations Of Proteins). Here, we describe the extension of COFFDROP to include bonded backbone terms derived from fitting to results of explicit-solvent MD simulations of all possible two-residue peptides containing the 20 standard amino acids, with histidine modeled in both its protonated and neutral forms. The iterative Boltzmann inversion (IBI) method was used to optimize new CG potential functions for backbone-related terms by attempting to reproduce angle, dihedral, and distance probability distributions generated by the MD simulations. In a simple test of the transferability of the extended force field, the angle, dihedral, and distance probability distributions obtained from BD simulations of 56 three-residue peptides were compared to results from corresponding explicit-solvent MD simulations. In a more challenging test of the COFFDROP force field, it was used to simulate eight intrinsically disordered proteins and was shown to quite accurately reproduce the experimental hydrodynamic radii (Rhydro), provided that the favorable nonbonded interactions of the force field were uniformly scaled downward in magnitude. Overall, the results indicate that the COFFDROP force field is likely to find use in modeling the conformational behavior of intrinsically disordered proteins and multidomain proteins connected by flexible linkers.

Ultrafast single molecule technique for the study of force dependent kinetics and conformational changes of actin-protein interaction involved in mechanotransduction

NASA Astrophysics Data System (ADS)

Sergides, M.; Arbore, C.; Pavone, F. S.; Capitanio, M.

2018-02-01

Mechanical signals occurring at the interface between cell membrane and extracellular matrix and at intercellular junctions trigger biochemical signals that are fundamental for cell growth, development and regulation. Adaptor proteins, which link the cell membrane to the actin cytoskeleton, seem to partake in this process of mechanotransduction. In particular, catenins play a key role in intercellular junctions, where they act as a bridge between the cell membrane and actin. Studies suggest that α-catenin contains a domain that normally masks vinculin binding sites, which can become accessible after a conformational change induced by an external force. Here we demonstrate a single-molecule technique for investigating actin-protein interactions at different forces (up to 17 pN) with adequate temporal resolution (sub-ms). This system is based on the ultrafast force-clamp spectroscopy technique that has been recently developed by our group and is adapted to study and measure force-dependent kinetics of the catenin-actin interaction, as well as the amplitude of the expected conformational changes such as force-induced protein unfolding.

A Generic Force Field for Protein Coarse-Grained Molecular Dynamics Simulation

PubMed Central

Gu, Junfeng; Bai, Fang; Li, Honglin; Wang, Xicheng

2012-01-01

Coarse-grained (CG) force fields have become promising tools for studies of protein behavior, but the balance of speed and accuracy is still a challenge in the research of protein coarse graining methodology. In this work, 20 CG beads have been designed based on the structures of amino acid residues, with which an amino acid can be represented by one or two beads, and a CG solvent model with five water molecules was adopted to ensure the consistence with the protein CG beads. The internal interactions in protein were classified according to the types of the interacting CG beads, and adequate potential functions were chosen and systematically parameterized to fit the energy distributions. The proposed CG force field has been tested on eight proteins, and each protein was simulated for 1000 ns. Even without any extra structure knowledge of the simulated proteins, the Cα root mean square deviations (RMSDs) with respect to their experimental structures are close to those of relatively short time all atom molecular dynamics simulations. However, our coarse grained force field will require further refinement to improve agreement with and persistence of native-like structures. In addition, the root mean square fluctuations (RMSFs) relative to the average structures derived from the simulations show that the conformational fluctuations of the proteins can be sampled. PMID:23203075

The Air Force Executive’s Guide on Stress: The Causes, Consequences, and Coping Strategies.

DTIC Science & Technology

1986-04-01

poultry, nuts, milk, yogurt , beef, pork, cheese, and eggs. Protein provides energy reserves and is used to grow and repair body tissue. (6:411 4513...EXERCISE WHY EXERCISE? Exercise is probably the most important means of becoming physically fit and buffering the effects of stress. Exercise which... buffer against stress. As a result, a person will experience less stress-induced illness, tension, anxiety, and depression. (6:190) 18 **-,:.-. N ~ "S

Colloid-probe AFM studies of the interaction forces of proteins adsorbed on colloidal crystals.

PubMed

Singh, Gurvinder; Bremmell, Kristen E; Griesser, Hans J; Kingshott, Peter

2015-04-28

In recent years, colloid-probe AFM has been used to measure the direct interaction forces between colloidal particles of different size or surface functionality in aqueous media, as one can study different forces in symmerical systems (i.e., sphere-sphere geometry). The present study investigates the interaction between protein coatings on colloid probes and hydrophilic surfaces decorated with hexagonally close packed single particle layers that are either uncoated or coated with proteins. Controlled solvent evaporation from aqueous suspensions of colloidal particles (coated with or without lysozyme and albumin) produces single layers of close-packed colloidal crystals over large areas on a solid support. The measurements have been carried out in an aqueous medium at different salt concentrations and pH values. The results show changes in the interaction forces as the surface charge of the unmodified or modified particles, and ionic strength or pH of the solution is altered. At high ionic strength or pH, electrostatic interactions are screened, and a strong repulsive force at short separation below 5 nm dominates, suggesting structural changes in the absorbed protein layer on the particles. We also study the force of adhesion, which decreases with an increment in the salt concentration, and the interaction between two different proteins indicating a repulsive interaction on approach and adhesion on retraction.

Binding preference of carbon nanotube over proline-rich motif ligand on SH3-domain: a comparison with different force fields.

PubMed

Shi, Biyun; Zuo, Guanghong; Xiu, Peng; Zhou, Ruhong

2013-04-04

With the widespread applications of nanomaterials such as carbon nanotubes, there is a growing concern on the biosafety of these engineered nanoparticles, in particular their interactions with proteins. In molecular simulations of nanoparticle-protein interactions, the choice of empirical parameters (force fields) plays a decisive role, and thus is of great importance and should be examined carefully before wider applications. Here we compare three commonly used force fields, CHARMM, OPLSAA, and AMBER in study of the competitive binding of a single wall carbon nanotube (SWCNT) with a native proline-rich motif (PRM) ligand on its target protein SH3 domain, a ubiquitous protein-protein interaction mediator involved in signaling and regulatory pathways. We find that the SWCNT displays a general preference over the PRM in binding with SH3 domain in all the three force fields examined, although the degree of preference can be somewhat different, with the AMBER force field showing the highest preference. The SWCNT prevents the ligand from reaching its native binding pocket by (i) occupying the binding pocket directly, and (ii) binding with the ligand itself and then being trapped together onto some off-sites. The π-π stacking interactions between the SWCNT and aromatic residues are found to play a significant role in its binding to the SH3 domain in all the three force fields. Further analyses show that even the SWCNT-ligand binding can also be relatively more stable than the native ligand-protein binding, indicating a serious potential disruption to the protein SH3 function.

CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca(2+)-ATPase.

PubMed

Komuro, Yasuaki; Re, Suyong; Kobayashi, Chigusa; Muneyuki, Eiro; Sugita, Yuji

2014-09-09

Adenosine triphosphate (ATP) is an indispensable energy source in cells. In a wide variety of biological phenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATP hydrolysis is converted to mechanical forces to bring about large-scale conformational changes in proteins. Investigation of structure-function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum Ca(2+)-ATPase and four soluble proteins. The ATP-bound structure of Ca(2+)-ATPase remains stable during MD simulations, contrary to the outcome in shorter simulations using original parameters. Similar results were obtained with the four ATP-bound soluble proteins. The new force-field parameters were also tested by investigating the range of conformations sampled during replica-exchange MD simulations of ATP in explicit water. Modified parameters allowed a much wider range of conformational sampling compared with the bias toward extended forms with original parameters. A diverse range of structures agrees with the broad distribution of ATP conformations in proteins deposited in the Protein Data Bank. These simulations suggest that the modified parameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins.

Optimization of the GBMV2 implicit solvent force field for accurate simulation of protein conformational equilibria.

PubMed

Lee, Kuo Hao; Chen, Jianhan

2017-06-15

Accurate treatment of solvent environment is critical for reliable simulations of protein conformational equilibria. Implicit treatment of solvation, such as using the generalized Born (GB) class of models arguably provides an optimal balance between computational efficiency and physical accuracy. Yet, GB models are frequently plagued by a tendency to generate overly compact structures. The physical origins of this drawback are relatively well understood, and the key to a balanced implicit solvent protein force field is careful optimization of physical parameters to achieve a sufficient level of cancellation of errors. The latter has been hampered by the difficulty of generating converged conformational ensembles of non-trivial model proteins using the popular replica exchange sampling technique. Here, we leverage improved sampling efficiency of a newly developed multi-scale enhanced sampling technique to re-optimize the generalized-Born with molecular volume (GBMV2) implicit solvent model with the CHARMM36 protein force field. Recursive optimization of key GBMV2 parameters (such as input radii) and protein torsion profiles (via the CMAP torsion cross terms) has led to a more balanced GBMV2 protein force field that recapitulates the structures and stabilities of both helical and β-hairpin model peptides. Importantly, this force field appears to be free of the over-compaction bias, and can generate structural ensembles of several intrinsically disordered proteins of various lengths that seem highly consistent with available experimental data. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Origins of structure in globular proteins.

PubMed Central

Chan, H S; Dill, K A

1990-01-01

The principal forces of protein folding--hydrophobicity and conformational entropy--are nonspecific. A long-standing puzzle has, therefore, been: What forces drive the formation of the specific internal architectures in globular proteins? We find that any self-avoiding flexible polymer molecule will develop large amounts of secondary structure, helices and parallel and antiparallel sheets, as it is driven to increasing compactness by any force of attraction among the chain monomers. Thus structure formation arises from the severity of steric constraints in compact polymers. This steric principle of organization can account for why short helices are stable in globular proteins, why there are parallel and anti-parallel sheets in proteins, and why weakly unfolded proteins have some secondary structure. On this basis, it should be possible to construct copolymers, not necessarily using amino acids, that can collapse to maximum compactness in incompatible solvents and that should then have structural organization resembling that of proteins. Images PMID:2385597

Parallel Tempering of Dark Matter from the Ebola Virus Proteome: Comparison of CHARMM36m and CHARMM22 Force Fields with Implicit Solvent.

PubMed

Olson, Mark A

2018-01-22

Intrinsically disordered proteins are characterized by their large manifold of thermally accessible conformations and their related statistical weights, making them an interesting target of simulation studies. To assess the development of a computational framework for modeling this distinct class of proteins, this work examines temperature-based replica-exchange simulations to generate a conformational ensemble of a 28-residue peptide from the Ebola virus protein VP35. Starting from a prefolded helix-β-turn-helix topology observed in a crystallographic assembly, the simulation strategy tested is the recently refined CHARMM36m force field combined with a generalized Born solvent model. A comparison of two replica-exchange methods is provided, where one is a traditional approach with a fixed set of temperatures and the other is an adaptive scheme in which the thermal windows are allowed to move in temperature space. The assessment is further extended to include a comparison with equivalent CHARMM22 simulation data sets. The analysis finds CHARMM36m to shift the minimum in the potential of mean force (PMF) to a lower fractional helicity compared with CHARMM22, while the latter showed greater conformational plasticity along the helix-forming reaction coordinate. Among the simulation models, only the adaptive tempering method with CHARMM36m found an ensemble of conformational heterogeneity consisting of transitions between α-helix-β-hairpin folds and unstructured states that produced a PMF of fractional fold propensity in qualitative agreement with circular dichroism experiments reporting a disordered peptide.

The Role of Non-Native Interactions in the Folding of Knotted Proteins: Insights from Molecular Dynamics Simulations

PubMed Central

Covino, Roberto; Škrbić, Tatjana; Beccara, Silvio a; Faccioli, Pietro; Micheletti, Cristian

2014-01-01

For several decades, the presence of knots in naturally-occurring proteins was largely ruled out a priori for its supposed incompatibility with the efficiency and robustness of folding processes. For this very same reason, the later discovery of several unrelated families of knotted proteins motivated researchers to look into the physico-chemical mechanisms governing the concerted sequence of folding steps leading to the consistent formation of the same knot type in the same protein location. Besides experiments, computational studies are providing considerable insight into these mechanisms. Here, we revisit a number of such recent investigations within a common conceptual and methodological framework. By considering studies employing protein models with different structural resolution (coarse-grained or atomistic) and various force fields (from pure native-centric to realistic atomistic ones), we focus on the role of native and non-native interactions. For various unrelated instances of knotted proteins, non-native interactions are shown to be very important for favoring the emergence of conformations primed for successful self-knotting events. PMID:24970203

The role of electrostatics in protein-protein interactions of a monoclonal antibody.

PubMed

Roberts, D; Keeling, R; Tracka, M; van der Walle, C F; Uddin, S; Warwicker, J; Curtis, R

2014-07-07

Understanding how protein-protein interactions depend on the choice of buffer, salt, ionic strength, and pH is needed to have better control over protein solution behavior. Here, we have characterized the pH and ionic strength dependence of protein-protein interactions in terms of an interaction parameter kD obtained from dynamic light scattering and the osmotic second virial coefficient B22 measured by static light scattering. A simplified protein-protein interaction model based on a Baxter adhesive potential and an electric double layer force is used to separate out the contributions of longer-ranged electrostatic interactions from short-ranged attractive forces. The ionic strength dependence of protein-protein interactions for solutions at pH 6.5 and below can be accurately captured using a Deryaguin-Landau-Verwey-Overbeek (DLVO) potential to describe the double layer forces. In solutions at pH 9, attractive electrostatics occur over the ionic strength range of 5-275 mM. At intermediate pH values (7.25 to 8.5), there is a crossover effect characterized by a nonmonotonic ionic strength dependence of protein-protein interactions, which can be rationalized by the competing effects of long-ranged repulsive double layer forces at low ionic strength and a shorter ranged electrostatic attraction, which dominates above a critical ionic strength. The change of interactions from repulsive to attractive indicates a concomitant change in the angular dependence of protein-protein interaction from isotropic to anisotropic. In the second part of the paper, we show how the Baxter adhesive potential can be used to predict values of kD from fitting to B22 measurements, thus providing a molecular basis for the linear correlation between the two protein-protein interaction parameters.

Extension of coarse-grained UNRES force field to treat carbon nanotubes.

PubMed

Sieradzan, Adam K; Mozolewska, Magdalena A

2018-04-26

Carbon nanotubes (CNTs) have recently received considerable attention because of their possible applications in various branches of nanotechnology. For their cogent application, knowledge of their interactions with biological macromolecules, especially proteins, is essential and computer simulations are very useful for such studies. Classical all-atom force fields limit simulation time scale and size of the systems significantly. Therefore, in this work, we implemented CNTs into the coarse-grained UNited RESidue (UNRES) force field. A CNT is represented as a rigid infinite-length cylinder which interacts with a protein through the Kihara potential. Energy conservation in microcanonical coarse-grained molecular dynamics simulations and temperature conservation in canonical simulations with UNRES containing the CNT component have been verified. Subsequently, studies of three proteins, bovine serum albumin (BSA), soybean peroxidase (SBP), and α-chymotrypsin (CT), with and without CNTs, were performed to examine the influence of CNTs on the structure and dynamics of these proteins. It was found that nanotubes bind to these proteins and influence their structure. Our results show that the UNRES force field can be used for further studies of CNT-protein systems with 3-4 order of magnitude larger timescale than using regular all-atom force fields. Graphical abstract Bovine serum albumin (BSA), soybean peroxidase (SBP), and α-chymotrypsin (CT), with and without CNTsᅟ.

Combination of atomic force microscopy and mass spectrometry for the detection of target protein in the serum samples of children with autism spectrum disorders

NASA Astrophysics Data System (ADS)

Kaysheva, A. L.; Pleshakova, T. O.; Kopylov, A. T.; Shumov, I. D.; Iourov, I. Y.; Vorsanova, S. G.; Yurov, Y. B.; Ziborov, V. S.; Archakov, A. I.; Ivanov, Y. D.

2017-10-01

Possibility of detection of target proteins associated with development of autistic disorders in children with use of combined atomic force microscopy and mass spectrometry (AFM/MS) method is demonstrated. The proposed method is based on the combination of affine enrichment of proteins from biological samples and visualization of these proteins by AFM and MS analysis with quantitative detection of target proteins.

Frustration in protein elastic network models

NASA Astrophysics Data System (ADS)

Lezon, Timothy; Bahar, Ivet

2010-03-01

Elastic network models (ENMs) are widely used for studying the equilibrium dynamics of proteins. The most common approach in ENM analysis is to adopt a uniform force constant or a non-specific distance dependent function to represent the force constant strength. Here we discuss the influence of sequence and structure in determining the effective force constants between residues in ENMs. Using a novel method based on entropy maximization, we optimize the force constants such that they exactly reporduce a subset of experimentally determined pair covariances for a set of proteins. We analyze the optimized force constants in terms of amino acid types, distances, contact order and secondary structure, and we demonstrate that including frustrated interactions in the ENM is essential for accurately reproducing the global modes in the middle of the frequency spectrum.

Design rules for biomolecular adhesion: lessons from force measurements.

PubMed

Leckband, Deborah

2010-01-01

Cell adhesion to matrix, other cells, or pathogens plays a pivotal role in many processes in biomolecular engineering. Early macroscopic methods of quantifying adhesion led to the development of quantitative models of cell adhesion and migration. The more recent use of sensitive probes to quantify the forces that alter or manipulate adhesion proteins has revealed much greater functional diversity than was apparent from population average measurements of cell adhesion. This review highlights theoretical and experimental methods that identified force-dependent molecular properties that are central to the biological activity of adhesion proteins. Experimental and theoretical methods emphasized in this review include the surface force apparatus, atomic force microscopy, and vesicle-based probes. Specific examples given illustrate how these tools have revealed unique properties of adhesion proteins and their structural origins.

AFM-based force spectroscopy on polystyrene brushes: effect of brush thickness on protein adsorption.

PubMed

Hentschel, Carsten; Wagner, Hendrik; Smiatek, Jens; Heuer, Andreas; Fuchs, Harald; Zhang, Xi; Studer, Armido; Chi, Lifeng

2013-02-12

Herein we present a study on nonspecific binding of proteins at highly dense packed hydrophobic polystyrene brushes. In this context, an atomic force microscopy tip was functionalized with concanavalin A to perform single-molecule force spectroscopy measurements on polystyrene brushes with thicknesses of 10 and 60 nm, respectively. Polystyrene brushes with thickness of 10 nm show an almost two times stronger protein adsorption than brushes with a thickness of 60 nm: 72 pN for the thinner and 38 pN for the thicker layer, which is in qualitative agreement with protein adsorption studies conducted macroscopically by fluorescence microscopy.

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair.

PubMed

LeBlanc, Sharonda; Wilkins, Hunter; Li, Zimeng; Kaur, Parminder; Wang, Hong; Erie, Dorothy A

2017-01-01

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein-protein and protein-DNA interactions. It can be used to capture snapshots of protein-DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein-protein and protein-DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein-protein and protein-DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein-DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein-protein and protein-nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure-function relationships that govern DNA repair processes. © 2017 Elsevier Inc. All rights reserved.

Electro-induced protein deposition on low-fouling surfaces

NASA Astrophysics Data System (ADS)

Cole, M. A.; Voelcker, N. H.; Thissen, H.

2007-12-01

Control over protein adsorption is a key issue for numerous biomedical applications ranging from diagnostic microarrays to tissue-engineered medical devices. Here, we describe a method for creating surfaces that prevent non-specific protein adsorption, which upon application of an external trigger can be transformed into surfaces showing high protein adsorption on demand. Silicon wafers were used as substrate materials upon which thin functional coatings were constructed by the deposition of an allylamine plasma polymer followed by high-density grafting of poly(ethylene oxide) aldehyde, resulting in a low-fouling surface. When the underlying highly doped silicon substrate was used as an electrode, the resulting electrostatic attraction between the electrode and charged proteins in solution induced protein deposition at the low-fouling interface. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were used to characterize the surface modifications. Controlled protein adsorption experiments were carried out using horseradish peroxidase. The amount of protein deposited at the surface was then investigated by means of a colorimetric assay. It is expected that the concept described here will find use in a variety of biotechnological and biomedical applications, particularly in the area of biochips.

Molecular Dynamics in Physiological Solutions: Force Fields, Alkali Metal Ions, and Ionic Strength.

PubMed

Zhang, Chao; Raugei, Simone; Eisenberg, Bob; Carloni, Paolo

2010-07-13

The monovalent ions Na(+) and K(+) and Cl(-) are present in any living organism. The fundamental thermodynamic properties of solutions containing such ions is given as the excess (electro-)chemical potential differences of single ions at finite ionic strength. This quantity is key for many biological processes, including ion permeation in membrane ion channels and DNA-protein interaction. It is given by a chemical contribution, related to the ion activity, and an electric contribution, related to the Galvani potential of the water/air interface. Here we investigate molecular dynamics based predictions of these quantities by using a variety of ion/water force fields commonly used in biological simulation, namely the AMBER (the newly developed), CHARMM, OPLS, Dang95 with TIP3P, and SPC/E water. Comparison with experiment is made with the corresponding values for salts, for which data are available. The calculations based on the newly developed AMBER force field with TIP3P water agrees well with experiment for both KCl and NaCl electrolytes in water solutions, as previously reported. The simulations based on the CHARMM-TIP3P and Dang95-SPC/E force fields agree well for the KCl and NaCl solutions, respectively. The other models are not as accurate. Single cations excess (electro-)chemical potential differences turn out to be similar for all the force fields considered here. In the case of KCl, the calculated electric contribution is consistent with higher level calculations. Instead, such agreement is not found with NaCl. Finally, we found that the calculated activities for single Cl(-) ions turn out to depend clearly on the type of counterion used, with all the force fields investigated. The implications of these findings for biomolecular systems are discussed.

Multiple stepwise refolding of immunoglobulin domain I27 upon force quench depends on initial conditions

PubMed Central

Li, Mai Suan; Hu, Chin-Kun; Klimov, Dmitri K.; Thirumalai, D.

2006-01-01

Mechanical folding trajectories for polyproteins starting from initially stretched conformations generated by single-molecule atomic force microscopy experiments [Fernandez, J. M. & Li, H. (2004) Science 303, 1674–1678] show that refolding, monitored by the end-to-end distance, occurs in distinct multiple stages. To clarify the molecular nature of folding starting from stretched conformations, we have probed the folding dynamics, upon force quench, for the single I27 domain from the muscle protein titin by using a Cα-Go model. Upon temperature quench, collapse and folding of I27 are synchronous. In contrast, refolding from stretched initial structures not only increases the folding and collapse time scales but also decouples the two kinetic processes. The increase in the folding times is associated primarily with the stretched state to compact random coil transition. Surprisingly, force quench does not alter the nature of the refolding kinetics, but merely increases the height of the free-energy folding barrier. Force quench refolding times scale as \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\tau}_{F}\\approx {\\tau}_{F}^{0}{\\mathrm{exp}}(f_{q}{\\Delta}x_{f}/k_{{\\mathrm{B}}}T)\\end{equation*}\\end{document}, where Δxf ≈ 0.6 nm is the location of the average transition state along the reaction coordinate given by end-to-end distance. We predict that τF and the folding mechanism can be dramatically altered by the initial and/or final values of force. The implications of our results for design and analysis of experiments are discussed. PMID:16373511

Importance of anisotropy in detachment rates for force production and cargo transport by a team of motor proteins.

PubMed

Takshak, Anjneya; Kunwar, Ambarish

2016-05-01

Many cellular processes are driven by collective forces generated by a team consisting of multiple molecular motor proteins. One aspect that has received less attention is the detachment rate of molecular motors under mechanical force/load. While detachment rate of kinesin motors measured under backward force increases rapidly for forces beyond stall-force; this scenario is just reversed for non-yeast dynein motors where detachment rate from microtubule decreases, exhibiting a catch-bond type behavior. It has been shown recently that yeast dynein responds anisotropically to applied load, i.e. detachment rates are different under forward and backward pulling. Here, we use computational modeling to show that these anisotropic detachment rates might help yeast dynein motors to improve their collective force generation in the absence of catch-bond behavior. We further show that the travel distance of cargos would be longer if detachment rates are anisotropic. Our results suggest that anisotropic detachment rates could be an alternative strategy for motors to improve the transport properties and force production by the team. © 2016 The Protein Society.

Neurochemical changes underlying schizophrenia-related behavior in a modified forced swim test in mice.

PubMed

Wożniak, Monika; Cieślik, Paulina; Marciniak, Marcin; Lenda, Tomasz; Pilc, Andrzej; Wieronska, Joanna M

2018-06-15

The modified forced swim test (MFST) has excellent predictive validity for investigating the antipsychotic activity of drugs, with particular emphasis on their activity toward negative symptoms of schizophrenia. However, its face and construct validity are less understood. Therefore, in the present study, some biochemical changes within GABAergic and serotonergic neurotransmission that could be related to observed MK-801-induced disturbances and the activity of compounds active at those neurotransmitters were investigated. In biochemical experiments, mice were treated acutely or chronically with MK-801 (13 days, 0.4 mg/kg). Their brains were dissected and frontal cortices and hippocampi were taken for further analysis. The levels of neurotransmitters were investigated with HPLC, and the expression of surrogate markers of schizophrenia (5-HT 1A receptors, GAD 65 , and GAD 67 , at both protein and mRNA levels) was measured via western blotting and q RT-PCR. The modified forced swim test and locomotor activity were used to assess the activity of GABA B and 5-HT 1A -related compounds. Repeated MK-801 treatment (13 days, 0.4 mg/kg dose) led to decreases in the DOPAC/DA, 3MT/DA and HVA/DA metabolic ratios. Increased 5-HT 1A protein expression and decreased GAD 65 and GAD 67 protein expression was observed in both the cortex and hippocampus. mRNA levels for all proteins were decreased. The increased immobility in the forced swim test was reversed both by a GABA B agonist (SKF97541, 0.025 or 0.05 mg/kg), a positive allosteric modulator of GABA B receptor (racBHFF, 5 or 10 mg/kg) and by a 5-HT 1A agonist ((R)-(+)-8-OH-DPAT 0.01 or 0.025 mg/kg). Our research supports the hypothesis that changes in the levels of GABA and/or 5-HT 1A receptors may contribute to the schizophrenia-like phenotype, and GABAergic and serotonergic agents may be good candidates for treating negative symptoms of schizophrenia. Copyright © 2018. Published by Elsevier Inc.

Pre-transition effects mediate forces of assembly between transmembrane proteins

DOE PAGES

Katira, Shachi; Mandadapu, Kranthi K.; Vaikuntanathan, Suriyanarayanan; ...

2016-02-24

We present a mechanism for a generic, powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the membrane. Using large-scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order–disorder interface. The stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial energy. Analogous to the hydrophobic effect, we refer to thismore » phenomenon as the 'orderphobic effect'. The effect is mediated by proximity to the order–disorder phase transition and the size and hydrophobic mismatch of the protein. The strength and range of forces arising from this effect are significantly larger than those that could arise from membrane elasticity for the membranes considered.« less

Pre-transition effects mediate forces of assembly between transmembrane proteins

PubMed Central

Katira, Shachi; Mandadapu, Kranthi K; Vaikuntanathan, Suriyanarayanan; Smit, Berend; Chandler, David

2016-01-01

We present a mechanism for a generic, powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the membrane. Using large-scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order–disorder interface. The stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial energy. Analogous to the hydrophobic effect, we refer to this phenomenon as the 'orderphobic effect'. The effect is mediated by proximity to the order–disorder phase transition and the size and hydrophobic mismatch of the protein. The strength and range of forces arising from this effect are significantly larger than those that could arise from membrane elasticity for the membranes considered. DOI: http://dx.doi.org/10.7554/eLife.13150.001 PMID:26910009

Pre-transition effects mediate forces of assembly between transmembrane proteins

DOE PAGES

Katira, Sachi; Mandadapu, Kranthi K.; Vaikuntanathan, Suriyanarayanan; ...

2016-02-24

We present a mechanism for a generic, powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the membrane. Using large-scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order-disorder interface. The stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial energy. Analogous to the hydrophobic effect, we refer to thismore » phenomenon as the ‘orderphobic effect’. The effect is mediated by proximity to the order-disorder phase transition and the size and hydrophobic mismatch of the protein. Furthermore, the strength and range of forces arising from this effect are significantly larger than those that could arise from membrane elasticity for the membranes considered.« less

Pre-transition effects mediate forces of assembly between transmembrane proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katira, Shachi; Mandadapu, Kranthi K.; Vaikuntanathan, Suriyanarayanan

We present a mechanism for a generic, powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the membrane. Using large-scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order–disorder interface. The stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial energy. Analogous to the hydrophobic effect, we refer to thismore » phenomenon as the 'orderphobic effect'. The effect is mediated by proximity to the order–disorder phase transition and the size and hydrophobic mismatch of the protein. The strength and range of forces arising from this effect are significantly larger than those that could arise from membrane elasticity for the membranes considered.« less

Pre-transition effects mediate forces of assembly between transmembrane proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katira, Sachi; Mandadapu, Kranthi K.; Vaikuntanathan, Suriyanarayanan

We present a mechanism for a generic, powerful force of assembly and mobility for transmembrane proteins in lipid bilayers. This force is a pre-transition (or pre-melting) effect for the first-order transition between ordered and disordered phases in the membrane. Using large-scale molecular simulation, we show that a protein with hydrophobic thickness equal to that of the disordered phase embedded in an ordered bilayer stabilizes a microscopic order-disorder interface. The stiffness of that interface is finite. When two such proteins approach each other, they assemble because assembly reduces the net interfacial energy. Analogous to the hydrophobic effect, we refer to thismore » phenomenon as the ‘orderphobic effect’. The effect is mediated by proximity to the order-disorder phase transition and the size and hydrophobic mismatch of the protein. Furthermore, the strength and range of forces arising from this effect are significantly larger than those that could arise from membrane elasticity for the membranes considered.« less

Diffusing colloidal probes of protein-carbohydrate interactions.

PubMed

Eichmann, Shannon L; Meric, Gulsum; Swavola, Julia C; Bevan, Michael A

2013-02-19

We present diffusing colloidal probe measurements of weak, multivalent, specific protein-polysaccharide interactions mediated by a competing monosaccharide. Specifically, we used integrated evanescent wave and video microscopy methods to monitor the three-dimensional Brownian excursions of conconavilin A (ConA) decorated colloids interacting with dextran-functionalized surfaces in the presence of glucose. Particle trajectories were interpreted as binding lifetime histograms, binding isotherms, and potentials of mean force. Binding lifetimes and isotherms showed clear trends of decreasing ConA-dextran-specific binding with increasing glucose concentration, consistent with expectations. Net potentials were accurately captured by superposition of a short-range, glucose-independent ConA-dextran repulsion and a longer-range, glucose-dependent dextran bridging attraction modeled as a harmonic potential. For glucose concentrations greater than 100 mM, the net ConA-dextran potential was found to have only a nonspecific repulsion, similar to that of bovine serum albumin (BSA) decorated colloids over dextran determined in control experiments. Our results demonstrate the first use of optical microscopy methods to quantify the connections between potentials of mean force and the binding behavior of ConA-decorated colloids on dextran-functionalized surfaces.

Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation.

PubMed

Lu, H; Isralewitz, B; Krammer, A; Vogel, V; Schulten, K

1998-08-01

Titin, a 1-microm-long protein found in striated muscle myofibrils, possesses unique elastic and extensibility properties in its I-band region, which is largely composed of a PEVK region (70% proline, glutamic acid, valine, and lysine residue) and seven-strand beta-sandwich immunoglobulin-like (Ig) domains. The behavior of titin as a multistage entropic spring has been shown in atomic force microscope and optical tweezer experiments to partially depend on the reversible unfolding of individual Ig domains. We performed steered molecular dynamics simulations to stretch single titin Ig domains in solution with pulling speeds of 0.5 and 1.0 A/ps. Resulting force-extension profiles exhibit a single dominant peak for each Ig domain unfolding, consistent with the experimentally observed sequential, as opposed to concerted, unfolding of Ig domains under external stretching forces. This force peak can be attributed to an initial burst of backbone hydrogen bonds, which takes place between antiparallel beta-strands A and B and between parallel beta-strands A' and G. Additional features of the simulations, including the position of the force peak and relative unfolding resistance of different Ig domains, can be related to experimental observations.

A Bioinspired Alginate-Gum Arabic Hydrogel with Micro-/Nanoscale Structures for Controlled Drug Release in Chronic Wound Healing.

PubMed

Li, Mi; Li, Haichang; Li, Xiangguang; Zhu, Hua; Xu, Zihui; Liu, Lianqing; Ma, Jianjie; Zhang, Mingjun

2017-07-12

Biopolymeric hydrogels have drawn increasing research interest in biomaterials due to their tunable physical and chemical properties for both creating bioactive cellular microenvironment and serving as sustainable therapeutic reagents. Inspired by a naturally occurring hydrogel secreted from the carnivorous Sundew plant for trapping insects, here we have developed a bioinspired hydrogel to deliver mitsugumin 53 (MG53), an important protein in cell membrane repair, for chronic wound healing. Both chemical compositions and micro-/nanomorphological properties inherent from the natural Sundew hydrogel were mimicked using sodium alginate and gum arabic with calcium ion-mediated cross-linking. On the basis of atomic force microscopy (AFM) force measurements, an optimal sticky hydrogel scaffold was obtained through orthogonal experimental design. Imaging and mechanical analysis showed the distinct correlation between structural morphology, adhesion characteristics, and mechanical properties of the Sundew-inspired hydrogel. Combined characterization and biochemistry techniques were utilized to uncover the underlying molecular composition involved in the interactions between hydrogel and protein. In vitro drug release experiments confirmed that the Sundew-inspired hydrogel had a biphasic-kinetics release, which can facilitate both fast delivery of MG53 for improving the reepithelization process of the wounds and sustained release of the protein for treating chronic wounds. In vivo experiments showed that the Sundew-inspired hydrogel encapsulating with rhMG53 could facilitate dermal wound healing in mouse model. Together, these studies confirmed that the Sundew-inspired hydrogel has both tunable micro-/nanostructures and physicochemical properties, which enable it as a delivery vehicle for chronic wounding healing. The research may provide a new way to develop biocompatible and tunable biomaterials for sustainable drug release to meet the needs of biological activities.

Optimizing Protein-Protein van der Waals Interactions for the AMBER ff9x/ff12 Force Field.

PubMed

Chapman, Dail E; Steck, Jonathan K; Nerenberg, Paul S

2014-01-14

The quality of molecular dynamics (MD) simulations relies heavily on the accuracy of the underlying force field. In recent years, considerable effort has been put into developing more accurate dihedral angle potentials for MD force fields, but relatively little work has focused on the nonbonded parameters, many of which are two decades old. In this work, we assess the accuracy of protein-protein van der Waals interactions in the AMBER ff9x/ff12 force field. Across a test set of 44 neat organic liquids containing the moieties present in proteins, we find root-mean-square (RMS) errors of 1.26 kcal/mol in enthalpy of vaporization and 0.36 g/cm(3) in liquid densities. We then optimize the van der Waals radii and well depths for all of the relevant atom types using these observables, which lowers the RMS errors in enthalpy of vaporization and liquid density of our validation set to 0.59 kcal/mol (53% reduction) and 0.019 g/cm(3) (46% reduction), respectively. Limitations in our parameter optimization were evident for certain atom types, however, and we discuss the implications of these observations for future force field development.

Parameterization of backbone flexibility in a coarse-grained force field for proteins (COFFDROP) derived from all-atom explicit-solvent molecular dynamics simulations of all possible two-residue peptides

PubMed Central

Frembgen-Kesner, Tamara; Andrews, Casey T.; Li, Shuxiang; Ngo, Nguyet Anh; Shubert, Scott A.; Jain, Aakash; Olayiwola, Oluwatoni; Weishaar, Mitch R.; Elcock, Adrian H.

2015-01-01

Recently, we reported the parameterization of a set of coarse-grained (CG) nonbonded potential functions, derived from all-atom explicit-solvent molecular dynamics (MD) simulations of amino acid pairs, and designed for use in (implicit-solvent) Brownian dynamics (BD) simulations of proteins; this force field was named COFFDROP (COarse-grained Force Field for Dynamic Representations Of Proteins). Here, we describe the extension of COFFDROP to include bonded backbone terms derived from fitting to results of explicit-solvent MD simulations of all possible two-residue peptides containing the 20 standard amino acids, with histidine modeled in both its protonated and neutral forms. The iterative Boltzmann inversion (IBI) method was used to optimize new CG potential functions for backbone-related terms by attempting to reproduce angle, dihedral and distance probability distributions generated by the MD simulations. In a simple test of the transferability of the extended force field, the angle, dihedral and distance probability distributions obtained from BD simulations of 56 three-residue peptides were compared to results from corresponding explicit-solvent MD simulations. In a more challenging test of the COFFDROP force field, it was used to simulate eight intrinsically disordered proteins and was shown to quite accurately reproduce the experimental hydrodynamic radii (Rhydro), provided that the favorable nonbonded interactions of the force field were uniformly scaled downwards in magnitude. Overall, the results indicate that the COFFDROP force field is likely to find use in modeling the conformational behavior of intrinsically disordered proteins and multi-domain proteins connected by flexible linkers. PMID:26574429

Viral nanomechanics with a virtual atomic force microscope

NASA Astrophysics Data System (ADS)

Aznar, María; Roca-Bonet, Sergi; Reguera, David

2018-07-01

One of the most important components of a virus is the protein shell or capsid that encloses its genetic material. The main role of the capsid is to protect the viral genome against external aggressions, facilitating its safe and efficient encapsulation and delivery. As a consequence, viral capsids have developed astonishing mechanical properties that are crucial for viral function. These remarkable properties have started to be unveiled in single-virus nanoindentation experiments, and are opening the door to the use of viral-derived artificial nanocages for promising bio- and nano-technological applications. However, the interpretation of nanoindentation experiments is often difficult, requiring the support of theoretical and simulation analysis. Here we present a ‘Virtual AFM’ (VAFM), a Brownian Dynamics simulation of a coarse-grained model of virus aimed to mimic the standard setup of atomic force microscopy (AFM) nanoindentation experiments. Despite the heavy level of coarse-graining, these simulations provide valuable information which is not accessible in experiments. Rather than focusing on a specific virus, the VAFM will be used to analyze how the mechanical response and breaking of viruses depend on different parameters controlling the effective interactions between capsid’s structural units. In particular, we will discuss the influence of adsorption, the tip radius, and the rigidity and shape of the shell on its mechanical response.

Origins of the Mechanochemical Coupling of Peptide Bond Formation to Protein Synthesis.

PubMed

Fritch, Benjamin; Kosolapov, Andrey; Hudson, Phillip; Nissley, Daniel A; Woodcock, H Lee; Deutsch, Carol; O'Brien, Edward P

2018-04-18

Mechanical forces acting on the ribosome can alter the speed of protein synthesis, indicating that mechanochemistry can contribute to translation control of gene expression. The naturally occurring sources of these mechanical forces, the mechanism by which they are transmitted 10 nm to the ribosome's catalytic core, and how they influence peptide bond formation rates are largely unknown. Here, we identify a new source of mechanical force acting on the ribosome by using in situ experimental measurements of changes in nascent-chain extension in the exit tunnel in conjunction with all-atom and coarse-grained computer simulations. We demonstrate that when the number of residues composing a nascent chain increases, its unstructured segments outside the ribosome exit tunnel generate piconewtons of force that are fully transmitted to the ribosome's P-site. The route of force transmission is shown to be through the nascent polypetide's backbone, not through the wall of the ribosome's exit tunnel. Utilizing quantum mechanical calculations we find that a consequence of such a pulling force is to decrease the transition state free energy barrier to peptide bond formation, indicating that the elongation of a nascent chain can accelerate translation. Since nascent protein segments can start out as largely unfolded structural ensembles, these results suggest a pulling force is present during protein synthesis that can modulate translation speed. The mechanism of force transmission we have identified and its consequences for peptide bond formation should be relevant regardless of the source of the pulling force.

Study of Fluid Flow Control In Protein Crystallization Using Strong Magnetic Fields

NASA Technical Reports Server (NTRS)

Ramachandran, N.; Leslie, F.; Ciszak, E.; Curreri, Peter A. (Technical Monitor)

2002-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in 'microgravity', researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined.

Study of Fluid Flow Control in Protein Crystallization using Strong Magnetic Fields

NASA Astrophysics Data System (ADS)

Ramachandran, Narayanan; Leslie, Fred; Ciszak, Ewa

2002-11-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in "microgravity", researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined. Specific goals for the investigation are: 1. To develop an understanding of convection control in diamagnetic fluids with concentration gradients through experimentation and numerical modeling. Specifically solutal buoyancy driven convection due to crystal growth will be considered. 2. To develop predictive measures for successful crystallization in a magnetic field using analyses and numerical modeling for use in future protein crystal growth experiments. This will establish criteria that can be used to estimate the efficacy of magnetic field flow damping on crystallization of candidate proteins. 3. To demonstrate the understanding of convection damping by high magnetic fields to a class of proteins that is of interest and whose structure is as yet not determined. 4. To compare quantitatively, the quality of the grown crystals with and without a magnetic field. X-ray diffraction techniques will be used for the comparative studies. In a preliminary set of experiments, we studied crystal dissolution effects in a 5 Tesla magnet available at NASA Marshall Space Flight Center (MSFC). Using a Schlieren setup, a 1mm crystal of Alum (Aluminum-Potassium Sulfate) was introduced in a 75% saturated solution and the resulting dissolution plume was observed. The experiment was conducted both in the presence and absence of a magnetic field gradient. The magnet produces a gradient field of approx. 1 Tesla2/cm. Image analysis of the recorded images indicated an enhanced plume velocity that was of the order of the measurement limit. For this experiment, both the gradient and gravity fields are in the same direction resulting in an enhanced effective gravity that tends to accelerate the observed plume velocity. While the results are not conclusive, pending further tests, it clearly points out the inadequacy of the MSFC magnet for conducting protein crystallization experiments and the need for a stronger magnet. In spacebased experiments, however, where the gravitational effects are small, only a weak magnetic field will be required to control or mitigate the effects of convective contamination.

Study of Fluid Flow Control in Protein Crystallization using Strong Magnetic Fields

NASA Technical Reports Server (NTRS)

Ramachandran, Narayanan; Leslie, Fred; Ciszak, Ewa

2002-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in "microgravity", researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined. Specific goals for the investigation are: 1. To develop an understanding of convection control in diamagnetic fluids with concentration gradients through experimentation and numerical modeling. Specifically solutal buoyancy driven convection due to crystal growth will be considered. 2. To develop predictive measures for successful crystallization in a magnetic field using analyses and numerical modeling for use in future protein crystal growth experiments. This will establish criteria that can be used to estimate the efficacy of magnetic field flow damping on crystallization of candidate proteins. 3. To demonstrate the understanding of convection damping by high magnetic fields to a class of proteins that is of interest and whose structure is as yet not determined. 4. To compare quantitatively, the quality of the grown crystals with and without a magnetic field. X-ray diffraction techniques will be used for the comparative studies. In a preliminary set of experiments, we studied crystal dissolution effects in a 5 Tesla magnet available at NASA Marshall Space Flight Center (MSFC). Using a Schlieren setup, a 1mm crystal of Alum (Aluminum-Potassium Sulfate) was introduced in a 75% saturated solution and the resulting dissolution plume was observed. The experiment was conducted both in the presence and absence of a magnetic field gradient. The magnet produces a gradient field of approx. 1 Tesla2/cm. Image analysis of the recorded images indicated an enhanced plume velocity that was of the order of the measurement limit. For this experiment, both the gradient and gravity fields are in the same direction resulting in an enhanced effective gravity that tends to accelerate the observed plume velocity. While the results are not conclusive, pending further tests, it clearly points out the inadequacy of the MSFC magnet for conducting protein crystallization experiments and the need for a stronger magnet. In spacebased experiments, however, where the gravitational effects are small, only a weak magnetic field will be required to control or mitigate the effects of convective contamination.

Optical Tweezers-Based Measurements of Forces and Dynamics at Microtubule Ends.

PubMed

Baclayon, Marian; Kalisch, Svenja-Marei; Hendel, Ed; Laan, Liedewij; Husson, Julien; Munteanu, E Laura; Dogterom, Marileen

2017-01-01

Microtubules are dynamic cytoskeletal polymers that polymerize and depolymerize while interacting with different proteins and structures within the cell. The highly regulated dynamic properties as well as the pushing and pulling forces generated by dynamic microtubule ends play important roles in processes such as in cell division. For instance, microtubule end-binding proteins are known to affect dramatically the dynamic properties of microtubules, and cortical dyneins are known to mediate pulling forces on microtubule ends. We discuss in this chapter our efforts to reconstitute these systems in vitro and mimic their interactions with structures within the cell using micro-fabricated barriers. Using an optical tweezers setup, we investigate the dynamics and forces of microtubules growing against functionalized barriers in the absence and presence of end-binding proteins and barrier-attached motor proteins. This setup allows high-speed as well as nanometer and piconewton resolution measurements on dynamic microtubules.

Toward structure prediction of cyclic peptides.

PubMed

Yu, Hongtao; Lin, Yu-Shan

2015-02-14

Cyclic peptides are a promising class of molecules that can be used to target specific protein-protein interactions. A computational method to accurately predict their structures would substantially advance the development of cyclic peptides as modulators of protein-protein interactions. Here, we develop a computational method that integrates bias-exchange metadynamics simulations, a Boltzmann reweighting scheme, dihedral principal component analysis and a modified density peak-based cluster analysis to provide a converged structural description for cyclic peptides. Using this method, we evaluate the performance of a number of popular protein force fields on a model cyclic peptide. All the tested force fields seem to over-stabilize the α-helix and PPII/β regions in the Ramachandran plot, commonly populated by linear peptides and proteins. Our findings suggest that re-parameterization of a force field that well describes the full Ramachandran plot is necessary to accurately model cyclic peptides.

Concerted dihedral rotations give rise to internal friction in unfolded proteins.

PubMed

Echeverria, Ignacia; Makarov, Dmitrii E; Papoian, Garegin A

2014-06-18

Protein chains undergo conformational diffusion during folding and dynamics, experiencing both thermal kicks and viscous drag. Recent experiments have shown that the corresponding friction can be separated into wet friction, which is determined by the solvent viscosity, and dry friction, where frictional effects arise due to the interactions within the protein chain. Despite important advances, the molecular origins underlying dry friction in proteins have remained unclear. To address this problem, we studied the dynamics of the unfolded cold-shock protein at different solvent viscosities and denaturant concentrations. Using extensive all-atom molecular dynamics simulations we estimated the internal friction time scales and found them to agree well with the corresponding experimental measurements (Soranno et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 17800-17806). Analysis of the reconfiguration dynamics of the unfolded chain further revealed that hops in the dihedral space provide the dominant mechanism of internal friction. Furthermore, the increased number of concerted dihedral moves at physiological conditions suggest that, in such conditions, the concerted motions result in higher frictional forces. These findings have important implications for understanding the folding kinetics of proteins as well as the dynamics of intrinsically disordered proteins.

Using Strong Magnetic Fields to Control Solutal Convection

NASA Technical Reports Server (NTRS)

Ramachandran, N.; Leslie, F. W.

2003-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in microgravity , we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the experimental results using paramagnetic salts and solutions in magnetic fields and compare them to analytical predictions.

Countering Solutal Buoyant Convection with High Magnetic Fields

NASA Technical Reports Server (NTRS)

Ramachandran, N.; Leslie, F. W.

2002-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemist, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitant, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in microgravity, we have been able to dramatically effect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the current status of the investigation and discuss results from the experimental and modeling efforts.

Evaluation of adhesion force between functionalized microbeads and protein-coated stainless steel using shear-flow-induced detachment.

PubMed

Mercier-Bonin, Muriel; Adoue, Mathieu; Zanna, Sandrine; Marcus, Philippe; Combes, Didier; Schmitz, Philippe

2009-10-01

Spherical microbeads functionalized with two types of chemical groups (NH(2), OH) were chosen as a simplified bacterial model, in order to elucidate the role of macromolecular interactions between specific biopolymers and 316 L stainless steel, in the frame of biofilm formation in the marine environment. NH(2) microbeads were used in their native form or after covalent binding to BSA or different representative poly-amino acids. OH microbeads were used in their native form. Adhesion force between microbeads and bare or BSA-coated stainless steel was quantified at nanoscale. Shear-flow-induced detachment experiments were combined with a simplified version of a theoretical model, based on the balance of hydrodynamic forces and torque exerted on microbeads. A maximal adhesion force of 27.6+/-8.5 nN was obtained for BSA-coated NH(2) microbeads. The high reactivity of OH functional groups was assessed (adhesion force of 15.6+/-4.8 nN for large microbeads). When charge-conducting stainless steel was coated with BSA, adhesion force was significantly lower than the one estimated with the bare surface, probably due to an increase in hydrophilic surface properties or suppression of charge transfer. The mechanism for microbead detachment was established (mainly rolling). The flow chamber and the associated theoretical modelling were demonstrated to be a relevant approach to quantify nanoscale forces between interacting surfaces.

Simulations Meet Experiment to Reveal New Insights into DNA Intrinsic Mechanics

PubMed Central

Ben Imeddourene, Akli; Elbahnsi, Ahmad; Guéroult, Marc; Oguey, Christophe; Foloppe, Nicolas; Hartmann, Brigitte

2015-01-01

The accurate prediction of the structure and dynamics of DNA remains a major challenge in computational biology due to the dearth of precise experimental information on DNA free in solution and limitations in the DNA force-fields underpinning the simulations. A new generation of force-fields has been developed to better represent the sequence-dependent B-DNA intrinsic mechanics, in particular with respect to the BI ↔ BII backbone equilibrium, which is essential to understand the B-DNA properties. Here, the performance of MD simulations with the newly updated force-fields Parmbsc0εζOLI and CHARMM36 was tested against a large ensemble of recent NMR data collected on four DNA dodecamers involved in nucleosome positioning. We find impressive progress towards a coherent, realistic representation of B-DNA in solution, despite residual shortcomings. This improved representation allows new and deeper interpretation of the experimental observables, including regarding the behavior of facing phosphate groups in complementary dinucleotides, and their modulation by the sequence. It also provides the opportunity to extensively revisit and refine the coupling between backbone states and inter base pair parameters, which emerges as a common theme across all the complementary dinucleotides. In sum, the global agreement between simulations and experiment reveals new aspects of intrinsic DNA mechanics, a key component of DNA-protein recognition. PMID:26657165

Surfing on Protein Waves: Proteophoresis as a Mechanism for Bacterial Genome Partitioning

NASA Astrophysics Data System (ADS)

Walter, J.-C.; Dorignac, J.; Lorman, V.; Rech, J.; Bouet, J.-Y.; Nollmann, M.; Palmeri, J.; Parmeggiani, A.; Geniet, F.

2017-07-01

Efficient bacterial chromosome segregation typically requires the coordinated action of a three-component machinery, fueled by adenosine triphosphate, called the partition complex. We present a phenomenological model accounting for the dynamic activity of this system that is also relevant for the physics of catalytic particles in active environments. The model is obtained by coupling simple linear reaction-diffusion equations with a proteophoresis, or "volumetric" chemophoresis, force field that arises from protein-protein interactions and provides a physically viable mechanism for complex translocation. This minimal description captures most known experimental observations: dynamic oscillations of complex components, complex separation, and subsequent symmetrical positioning. The predictions of our model are in phenomenological agreement with and provide substantial insight into recent experiments. From a nonlinear physics view point, this system explores the active separation of matter at micrometric scales with a dynamical instability between static positioning and traveling wave regimes triggered by the dynamical spontaneous breaking of rotational symmetry.

“Ultra-high resolution optical trap with single fluorophore sensitivity”

PubMed Central

Comstock, Matthew J; Ha, Taekjip; Chemla, Yann R

2013-01-01

We present a single-molecule instrument that combines a timeshared ultra-high resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, individual single-fluorophore labeled DNA oligonucleotides were observed to bind and unbind to complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, coincident angstrom-scale changes in tether extension could be clearly observed. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (e.g., single base pair stepping of DNA translocases) along with the detection of fluorescently labeled protein properties (e.g., internal configuration). PMID:21336286

Fluctuations in protein synthesis from a single RNA template: stochastic kinetics of ribosomes.

PubMed

Garai, Ashok; Chowdhury, Debashish; Ramakrishnan, T V

2009-01-01

Proteins are polymerized by cyclic machines called ribosomes, which use their messenger RNA (mRNA) track also as the corresponding template, and the process is called translation. We explore, in depth and detail, the stochastic nature of the translation. We compute various distributions associated with the translation process; one of them--namely, the dwell time distribution--has been measured in recent single-ribosome experiments. The form of the distribution, which fits best with our simulation data, is consistent with that extracted from the experimental data. For our computations, we use a model that captures both the mechanochemistry of each individual ribosome and their steric interactions. We also demonstrate the effects of the sequence inhomogeneities of real genes on the fluctuations and noise in translation. Finally, inspired by recent advances in the experimental techniques of manipulating single ribosomes, we make theoretical predictions on the force-velocity relation for individual ribosomes. In principle, all our predictions can be tested by carrying out in vitro experiments.

The Experience of Force: The Role of Haptic Experience of Forces in Visual Perception of Object Motion and Interactions, Mental Simulation, and Motion-Related Judgments

ERIC Educational Resources Information Center

White, Peter A.

2012-01-01

Forces are experienced in actions on objects. The mechanoreceptor system is stimulated by proximal forces in interactions with objects, and experiences of force occur in a context of information yielded by other sensory modalities, principally vision. These experiences are registered and stored as episodic traces in the brain. These stored…

Improving single molecule force spectroscopy through automated real-time data collection and quantification of experimental conditions

PubMed Central

Scholl, Zackary N.; Marszalek, Piotr E.

2013-01-01

The benefits of single molecule force spectroscopy (SMFS) clearly outweigh the challenges which include small sample sizes, tedious data collection and introduction of human bias during the subjective data selection. These difficulties can be partially eliminated through automation of the experimental data collection process for atomic force microscopy (AFM). Automation can be accomplished using an algorithm that triages usable force-extension recordings quickly with positive and negative selection. We implemented an algorithm based on the windowed fast Fourier transform of force-extension traces that identifies peaks using force-extension regimes to correctly identify usable recordings from proteins composed of repeated domains. This algorithm excels as a real-time diagnostic because it involves <30 ms computational time, has high sensitivity and specificity, and efficiently detects weak unfolding events. We used the statistics provided by the automated procedure to clearly demonstrate the properties of molecular adhesion and how these properties change with differences in the cantilever tip and protein functional groups and protein age. PMID:24001740

The mapping of yeast's G-protein coupled receptor with an atomic force microscope

NASA Astrophysics Data System (ADS)

Takenaka, Musashi; Miyachi, Yusuke; Ishii, Jun; Ogino, Chiaki; Kondo, Akihiko

2015-03-01

An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, α-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations.

PubMed

Kukic, Predrag; Kannan, Arvind; Dijkstra, Maurits J J; Abeln, Sanne; Camilloni, Carlo; Vendruscolo, Michele

2015-10-01

It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a 'tube model' approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the 'CamTube' force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 μs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost.

Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations

PubMed Central

Dijkstra, Maurits J. J.; Abeln, Sanne; Camilloni, Carlo; Vendruscolo, Michele

2015-01-01

It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a ‘tube model’ approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the ‘CamTube’ force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 μs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost. PMID:26505754

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

PubMed Central

Thoren, Katie L.; Worden, Evan J.; Yassif, Jaime M.; Krantz, Bryan A.

2009-01-01

Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal β-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines. PMID:19926859

Nanoscale Electron Transport Measurements of Immobilized Cytochrome P450 Proteins

PubMed Central

Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

2015-01-01

Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. PMID:25804257

Development And Use Of Advanced Microfabricated Traction Force Sensing Substrates To Study The Effect of Nanosilver On Human Macrophages

NASA Astrophysics Data System (ADS)

Stark, Daniel Thomas

While nanoparticles are a natural byproduct of combustion and a number of natural processes, engineered nanoparticles have only recently entered the consumer market. This motivates the development of methods for studying their effects on human cells, thereby indicating how larger models such as animals and humans might react to them. This research develops a method to mechanically characterize cellular traction forces as a measure of exposure to nanoparticles. To do this, 1microm micropillar molds were fabricated in silicon wafers using smooth sidewall reactive ion plasma etching technologies. Polydimethylsiloxane (PDMS), was cured inside the silicon molds, subsequently treated for cell culture and used to measure cellular traction forces over time in live cell time-lapse experiments. For the first time, transmitted light was used to visualize the PDMS micropillars; a force resolution of 5.6 +/-2.1nN was achieved across all experiments using a standard Olympus IX81 confocal microscope affixed with a 60x NA2.1 objective. To initiate cellular movement, monocyte chemoattractant protein (MCP-1) was conjugated to 1microm latex beads. The effects of 40nm silver nanoparticle exposures were quantified using the micropillar array. Changes in cellular behavior between the control group and cells exposed to nanosilver were not significant, although a comparison between the 5microg/ml and 10microg/ml nanosilver concentrations yielded strong significance using a 2 sided Students t test.

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer's disease.

PubMed

Grégoire, C; Marco, S; Thimonier, J; Duplan, L; Laurine, E; Chauvin, J P; Michel, B; Peyrot, V; Verdier, J M

2001-07-02

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre-clinical stages of Alzheimer's disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimer's disease. After self-proteolysis, the N-terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three-dimensional X-ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple-helical filament (QHF-litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.

ff14ipq: A Self-Consistent Force Field for Condensed-Phase Simulations of Proteins

PubMed Central

2015-01-01

We present the ff14ipq force field, implementing the previously published IPolQ charge set for simulations of complete proteins. Minor modifications to the charge derivation scheme and van der Waals interactions between polar atoms are introduced. Torsion parameters are developed through a generational learning approach, based on gas-phase MP2/cc-pVTZ single-point energies computed of structures optimized by the force field itself rather than the quantum benchmark. In this manner, we sacrifice information about the true quantum minima in order to ensure that the force field maintains optimal agreement with the MP2/cc-pVTZ benchmark for the ensembles it will actually produce in simulations. A means of making the gas-phase torsion parameters compatible with solution-phase IPolQ charges is presented. The ff14ipq model is an alternative to ff99SB and other Amber force fields for protein simulations in programs that accommodate pair-specific Lennard–Jones combining rules. The force field gives strong performance on α-helical and β-sheet oligopeptides as well as globular proteins over microsecond time scale simulations, although it has not yet been tested in conjunction with lipid and nucleic acid models. We show how our choices in parameter development influence the resulting force field and how other choices that may have appeared reasonable would actually have led to poorer results. The tools we developed may also aid in the development of future fixed-charge and even polarizable biomolecular force fields. PMID:25328495

Rate limit of protein elastic response is tether dependent.

PubMed

Berkovich, Ronen; Hermans, Rodolfo I; Popa, Ionel; Stirnemann, Guillaume; Garcia-Manyes, Sergi; Berne, Bruce J; Fernandez, Julio M

2012-09-04

The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient D(eff) ~ 1,200 nm(2)/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at D(eff) ~ 10(8) nm(2)/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo with D(eff) ~ 10(4)-10(6) nm(2)/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk.

PROTEIN METABOLISM AND EXCHANGE AS INFLUENCED BY CONSTRICTION OF THE VENA CAVA

PubMed Central

McKee, Frank W.; Hyatt, Robert E.; Wilt, William G.; Tishkoff, Garson H.; Whipple, George H.

1949-01-01

Further studies of ascitic fluid production and related factors in dogs with constriction of the vena cava above the diaphragm are reported. Whole dog plasma given intravenously to such animals produces a rise in circulating plasma protein to normal levels, but increases the output of ascitic fluid with a loss of protein via the ascites equivalent to 72, 76, and 65 per cent respectively, of the injected protein. Forced ingestion of water in excess of the test animal's normal needs and desires produces no significant changes in the circulating plasma protein level or in ascitic fluid production. Amino acid growth mixtures given intravenously in distilled water cause weight loss, elevation of circulating plasma proteins, a slightly negative nitrogen balance, but no ascitic fluid production. Amino acid growth mixtures given intravenously in normal saline cause depression of the circulating plasma proteins, negative nitrogen balance, and significant ascitic fluid production. Ascitic fluid given intravenously to the test animals causes a marked depression of circulating plasma proteins, a marked increase in ascitic fluid production containing the equivalent of 116 and 98 per cent of the injected protein, and a negative nitrogen balance. Ascitic fluid given orally produces a marked depression of circulating plasma proteins, and a marked increase in ascitic fluid secretion, containing the equivalent of 66, 66, and 54 per cent respectively, of the ingested protein. Sodium chloride is a dominant factor in some of these experiments where abundant ascites production is recorded. Protein levels and intake are important, but take second place to sodium. Ascitic fluids show electrophoretic patterns which are almost identical to the plasma patterns. The A/G ratios are often equal in ascitic fluid and plasma, sometimes even lower in the ascitic fluid. This emphasizes the ease with which globulins pass cell or other membrane barriers in these experiments. PMID:18143588

Forced Unfolding of Proteins Within Cells

PubMed Central

Johnson, Colin P.; Tang, Hsin-Yao; Carag, Christine; Speicher, David W.; Discher, Dennis E.

2009-01-01

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells—as a prototypical adherent cell—nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to colocalize signaling events such as phosphorylation with forced unfolding. PMID:17673662

Modification and optimization of the united-residue (UNRES) potential-energy function for canonical simulations. I. Temperature dependence of the effective energy function and tests of the optimization method with single training proteins

PubMed Central

Liwo, Adam; Khalili, Mey; Czaplewski, Cezary; Kalinowski, Sebastian; Ołdziej, Stanisław; Wachucik, Katarzyna; Scheraga, Harold A.

2011-01-01

We report the modification and parameterization of the united-residue (UNRES) force field for energy-based protein-structure prediction and protein-folding simulations. We tested the approach on three training proteins separately: 1E0L (β), 1GAB (α), and 1E0G (α + β). Heretofore, the UNRES force field had been designed and parameterized to locate native-like structures of proteins as global minima of their effective potential-energy surfaces, which largely neglected the conformational entropy because decoys composed of only lowest-energy conformations were used to optimize the force field. Recently, we developed a mesoscopic dynamics procedure for UNRES, and applied it with success to simulate protein folding pathways. How ever, the force field turned out to be largely biased towards α-helical structures in canonical simulations because the conformational entropy had been neglected in the parameterization. We applied the hierarchical optimization method developed in our earlier work to optimize the force field, in which the conformational space of a training protein is divided into levels each corresponding to a certain degree of native-likeness. The levels are ordered according to increasing native-likeness; level 0 corresponds to structures with no native-like elements and the highest level corresponds to the fully native-like structures. The aim of optimization is to achieve the order of the free energies of levels, decreasing as their native-likeness increases. The procedure is iterative, and decoys of the training protein(s) generated with the energy-function parameters of the preceding iteration are used to optimize the force field in a current iteration. We applied the multiplexing replica exchange molecular dynamics (MREMD) method, recently implemented in UNRES, to generate decoys; with this modification, conformational entropy is taken into account. Moreover, we optimized the free-energy gaps between levels at temperatures corresponding to a predominance of folded or unfolded structures, as well as to structures at the putative folding-transition temperature, changing the sign of the gaps at the transition temperature. This enabled us to obtain force fields characterized by a single peak in the heat capacity at the transition temperature. Furthermore, we introduced temperature dependence to the UNRES force field; this is consistent with the fact that it is a free-energy and not a potential-energy function. PMID:17201450

Elastic Molecular Machines and a New Motive Force in Protein Mechanisms,

DTIC Science & Technology

1987-01-01

coupling of the second kind. It is proposed that these new considerations are relevant to mechanisms for the turning on and off of elastic forces in protein mechanisms as varied as those of enzymes and muscle contraction .

Random myosin loss along thick-filaments increases myosin attachment time and the proportion of bound myosin heads to mitigate force decline in skeletal muscle

PubMed Central

Tanner, Bertrand C.W.; McNabb, Mark; Palmer, Bradley M.; Toth, Michael J.; Miller, Mark S.

2014-01-01

Diminished skeletal muscle performance with aging, disuse, and disease may be partially attributed to the loss of myofilament proteins. Several laboratories have found a disproportionate loss of myosin protein content relative to other myofilament proteins, but due to methodological limitations, the structural manifestation of this protein loss is unknown. To investigate how variations in myosin content affect ensemble cross-bridge behavior and force production we simulated muscle contraction in the half-sarcomere as myosin was removed either i) uniformly, from the Z-line end of thick-filaments, or ii) randomly, along the length of thick-filaments. Uniform myosin removal decreased force production, showing a slightly steeper force-to-myosin content relationship than the 1:1 relationship that would be expected from the loss of cross-bridges. Random myosin removal also decreased force production, but this decrease was less than observed with uniform myosin loss, largely due to increased myosin attachment time (ton) and fractional cross-bridge binding with random myosin loss. These findings support our prior observations that prolonged ton may augment force production in single fibers with randomly reduced myosin content from chronic heart failure patients. These simulation also illustrate that the pattern of myosin loss along thick-filaments influences ensemble cross-bridge behavior and maintenance of force throughout the sarcomere. PMID:24486373

Force Exertion and Transmission in Cross-Linked Actin Networks

NASA Astrophysics Data System (ADS)

Stam, Samantha

Cells are responsive to external cues in their environment telling them to proliferate or migrate within their surrounding tissue. Sensing of cues that are mechanical in nature, such stiffness of a tissue or forces transmitted from other cells, is believed to involve the cytoskeleton of a cell. The cytoskeleton is a complex network of proteins consisting of polymers that provide structural support, motor proteins that remodel these structures, and many others. We do not yet have a complete understanding of how cytoskeletal components respond to either internal or external mechanical force and stiffness. Such an understanding should involve mechanisms by which constituent molecules, such as motor proteins, are responsive to mechanics. Additionally, physical models of how forces are transmitted through biopolymer networks are necessary. My research has focused on networks formed by the cytoskeletal filament actin and the molecular motor protein myosin II. Actin filaments form networks and bundles that form a structural framework of the cell, and myosin II slides actin filaments. In this thesis, we show that stiffness of an elastic load that opposes myosin-generated actin sliding has a very sharp effect on the myosin force output in simulations. Secondly, we show that the stiffness and connectivity of cytoskeletal filaments regulates the contractility and anisotropy of network deformations that transmit force on material length scales. Together, these results have implications for predicting and interpreting the deformations and forces in biopolymeric active materials.

Single-molecule force measurement via optical tweezers reveals different kinetic features of two BRaf mutants responsible for cardio-facial-cutaneous (CFC) syndrome

PubMed Central

Wen, Cheng; Ye, Anpei

2013-01-01

BRaf (B- Rapid Accelerated Fibrosarcoma) protein is an important serine/threonine-protein kinase. Two domains on BRaf can independently bind its upstream kinase, Ras (Rat Sarcoma) protein. These are the Ras binding domain (RBD) and cysteine-rich-domain (CRD). Herein we use customized optical tweezers to compare the Ras binding process in two pathological mutants of BRaf responsible for CFC syndrome, abbreviated BRaf (A246P) and BRaf (Q257R). The two mutants differ in their kinetics of Ras-binding, though both bind Ras with similar increased overall affinity. BRaf (A246P) exhibits a slightly higher Ras/CRD unbinding force and a significantly higher Ras/RBD unbinding force versus the wild type. The contrary phenomenon is observed in the Q257R mutation. Simulations of the unstressed-off rate, koff(0), yield results in accordance with the changes revealed by the mean unbinding force. Our approach can be applied to rapidly assess other mutated proteins to deduce the effects of mutation on their kinetics compared to wild type proteins and to each other. PMID:24409384

Fast and Forceful Refolding of Stretched α-Helical Solenoid Proteins

PubMed Central

Kim, Minkyu; Abdi, Khadar; Lee, Gwangrog; Rabbi, Mahir; Lee, Whasil; Yang, Ming; Schofield, Christopher J.; Bennett, Vann; Marszalek, Piotr E.

2010-01-01

Abstract Anfinsen's thermodynamic hypothesis implies that proteins can encode for stretching through reversible loss of structure. However, large in vitro extensions of proteins that occur through a progressive unfolding of their domains typically dissipate a significant amount of energy, and therefore are not thermodynamically reversible. Some coiled-coil proteins have been found to stretch nearly reversibly, although their extension is typically limited to 2.5 times their folded length. Here, we report investigations on the mechanical properties of individual molecules of ankyrin-R, β-catenin, and clathrin, which are representative examples of over 800 predicted human proteins composed of tightly packed α-helical repeats (termed ANK, ARM, or HEAT repeats, respectively) that form spiral-shaped protein domains. Using atomic force spectroscopy, we find that these polypeptides possess unprecedented stretch ratios on the order of 10–15, exceeding that of other proteins studied so far, and their extension and relaxation occurs with minimal energy dissipation. Their sequence-encoded elasticity is governed by stepwise unfolding of small repeats, which upon relaxation of the stretching force rapidly and forcefully refold, minimizing the hysteresis between the stretching and relaxing parts of the cycle. Thus, we identify a new class of proteins that behave as highly reversible nanosprings that have the potential to function as mechanosensors in cells and as building blocks in springy nanostructures. Our physical view of the protein component of cells as being comprised of predominantly inextensible structural elements under tension may need revision to incorporate springs. PMID:20550922

Force transduction and lipid binding in MscL: A continuum-molecular approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanegas, Juan M.; Arroyo, Marino; Fotiadis, Dimitrios

2014-12-01

The bacterial mechanosensitive channel MscL, a small protein mainly activated by membrane tension, is a central model system to study the transduction of mechanical stimuli into chemical signals. Mutagenic studies suggest that MscL gating strongly depends on both intra-protein and interfacial lipid-protein interactions. However, there is a gap between this detailed chemical information and current mechanical models of MscL gating. Here, we investigate the MscL bilayer-protein interface through molecular dynamics simulations, and take a combined continuum-molecular approach to connect chemistry and mechanics. We quantify the effect of membrane tension on the forces acting on the surface of the channel, andmore » identify interactions that may be critical in the force transduction between the membrane and MscL. We find that the local stress distribution on the protein surface is largely asymmetric, particularly under tension, with the cytoplasmic side showing significantly larger and more localized forces, which pull the protein radially outward. The molecular interactions that mediate this behavior arise from hydrogen bonds between the electronegative oxygens in the lipid headgroup and a cluster of positively charged lysine residues on the amphipathic S1 domain and the C-terminal end of the second trans-membrane helix. We take advantage of this strong interaction (estimated to be 10–13 kT per lipid) to actuate the channel (by applying forces on protein-bound lipids) and explore its sensitivity to the pulling magnitude and direction. We conclude by highlighting the simple motif that confers MscL with strong anchoring to the bilayer, and its presence in various integral membrane proteins including the human mechanosensitive channel K2P1 and bovine rhodopsin.« less

Nature and consequences of protein-protein interactions in high protein concentration solutions.

PubMed

Saluja, Atul; Kalonia, Devendra S

2008-06-24

High protein concentration solutions are becoming increasingly important in the pharmaceutical industry. The solution behavior of proteins at high concentrations can markedly differ from that predicted based on dilute solution analysis due to thermodynamic non-ideality in these solutions. The non-ideality observed in these systems is related to the protein-protein interactions (PPI). Different types of forces play a key role in determining the overall nature and extent of these PPI and their relative contributions are affected by solute and solvent properties. However, individual contributions of these forces to the solution properties of concentrated protein solutions are not fully understood. The role of PPI, driven by these intermolecular forces, in governing solution rheology and physical stability of high protein concentration solutions is discussed from the point of view of pharmaceutical product development. Investigation of protein self-association and aggregation in concentrated protein solutions is crucial for ensuring the safety and efficacy of the final product for the duration of the desired product shelf life. Understanding rheology of high concentration protein solutions is critical for addressing issues during product manufacture and administration of final formulation to the patient. To this end, analysis of solution viscoelastic character can also provide an insight into the nature of PPI affecting solution rheology.

Molecular Dynamics Simulations, Challenges and Opportunities: A Biologist's Prospective.

PubMed

Kumari, Indu; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

2017-08-30

Molecular dynamics (MD) is a computational technique which is used to study biomolecules in virtual environment. Each of the constituent atoms represents a particle and hence the biomolecule embodies a multi-particle mechanical system analyzed within a simulation box during MD analysis. The potential energies of the atoms are explained by a mathematical expression consisting of different forces and space parameters. There are various software and force fields that have been developed for MD studies of the biomolecules. MD analysis has unravelled the various biological mechanisms (protein folding/unfolding, protein-small molecule interactions, protein-protein interactions, DNA/RNA-protein interactions, proteins embedded in membrane, lipid-lipid interactions, drug transport etc.) operating at the atomic and molecular levels. However, there are still some parameters including torsions in amino acids, carbohydrates (whose structure is extended and not well defined like that of proteins) and single stranded nucleic acids for which the force fields need further improvement, although there are several workers putting in constant efforts in these directions. The existing force fields are not efficient for studying the crowded environment inside the cells, since these interactions involve multiple factors in real time. Therefore, the improved force fields may provide the opportunities for their wider applications on the complex biosystems in diverse cellular conditions. In conclusion, the intervention of MD in the basic sciences involving interdisciplinary approaches will be helpful for understanding many fundamental biological and physiological processes at the molecular levels that may be further applied in various fields including biotechnology, fisheries, sustainable agriculture and biomedical research. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Space Shuttle Projects

NASA Image and Video Library

1991-04-05

Aboard the Space Shuttle Atlantis, the STS-37 mission launched April 5, 1991 from launch pad 39B at the Kennedy Space Center in Florida, and landed back on Earth April 11, 1991. The 39th shuttle mission included crew members: Steven R. Nagel, commander; Kenneth D. Cameron, pilot; Jerry L,. Ross, mission specialist 1; Jay Apt, mission specialist 2; and Linda M. Godwin, mission specialist 3. The primary payload for the mission was the Gamma Ray Observatory (GRO). The GRO included the Burst and Transient Experiment (BATSE); the Imaging Compton Telescope (COMPTEL); the Energetic Gamma Ray Experiment Telescope (EGRET); and the Oriented Scintillation Spectrometer Experiment (OSSEE). Secondary payloads included Crew and Equipment Translation Aids (CETA); the Ascent Particle Monitor (APM); the Shuttle Amateur Radio Experiment II (SAREXII), the Protein Crystal Growth (PCG); the Bioserve Instrumentation Technology Associates Materials Dispersion Apparatus (BIMDA); Radiation Monitoring Equipment III (RMEIII); and Air Force Maui Optical Site (AMOS).

Extracting Models in Single Molecule Experiments

NASA Astrophysics Data System (ADS)

Presse, Steve

2013-03-01

Single molecule experiments can now monitor the journey of a protein from its assembly near a ribosome to its proteolytic demise. Ideally all single molecule data should be self-explanatory. However data originating from single molecule experiments is particularly challenging to interpret on account of fluctuations and noise at such small scales. Realistically, basic understanding comes from models carefully extracted from the noisy data. Statistical mechanics, and maximum entropy in particular, provide a powerful framework for accomplishing this task in a principled fashion. Here I will discuss our work in extracting conformational memory from single molecule force spectroscopy experiments on large biomolecules. One clear advantage of this method is that we let the data tend towards the correct model, we do not fit the data. I will show that the dynamical model of the single molecule dynamics which emerges from this analysis is often more textured and complex than could otherwise come from fitting the data to a pre-conceived model.

In situ observation of fluoride-ion-induced hydroxyapatite collagen detachment on bone fracture surfaces by atomic force microscopy

NASA Astrophysics Data System (ADS)

Kindt, J. H.; Thurner, P. J.; Lauer, M. E.; Bosma, B. L.; Schitter, G.; Fantner, G. E.; Izumi, M.; Weaver, J. C.; Morse, D. E.; Hansma, P. K.

2007-04-01

The topography of freshly fractured bovine and human bone surfaces was determined by the use of atomic force microscopy (AFM). Fracture surfaces from both kinds of samples exhibited complex landscapes formed by hydroxyapatite mineral platelets with lateral dimensions ranging from ~90 nm × 60 nm to ~20 nm × 20 nm. Novel AFM techniques were used to study these fracture surfaces during various chemical treatments. Significant topographical changes were observed following exposure to aqueous solutions of ethylenediaminetetraacetic acid (EDTA) or highly concentrated sodium fluoride (NaF). Both treatments resulted in the apparent loss of the hydroxyapatite mineral platelets on a timescale of a few seconds. Collagen fibrils situated beneath the overlying mineral platelets were clearly exposed and could be resolved with high spatial resolution in the acquired AFM images. Time-dependent mass loss experiments revealed that the applied agents (NaF or EDTA) had very different resulting effects. Despite the fact that the two treatments exhibited nearly identical results following examination by AFM, bulk bone samples treated with EDTA exhibited a ~70% mass loss after 72 h, whereas for the NaF-treated samples, the mass loss was only of the order of ~10%. These results support those obtained from previous mechanical testing experiments, suggesting that enhanced formation of superficial fluoroapatite dramatically weakens the protein-hydroxyapatite interfaces. Additionally, we discovered that treatment with aqueous solutions of NaF resulted in the effective extraction of noncollagenous proteins from bone powder.

Magnetic Control of Convection during Protein Crystallization

NASA Technical Reports Server (NTRS)

Ramachandran, N.; Leslie, F. W.

2004-01-01

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular Crystals for diffraction analyses has been the central focus for bio-chemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and Sedimentation as is achieved in "microgravity", we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, f o d o n of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with counteracts on for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteract terrestrial gravity. The genera1 objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the experimental results using paramagentic salts and solutions in magnetic fields and compare them to analyticalprctions.

Advanced imaging microscope tools applied to microgravity research investigations

NASA Astrophysics Data System (ADS)

Peterson, L.; Samson, J.; Conrad, D.; Clark, K.

1998-01-01

The inability to observe and interact with experiments on orbit has been an impediment for both basic research and commercial ventures using the shuttle. In order to open the frontiers of space, the Center for Microgravity Automation Technology has developed a unique and innovative system for conducting experiments at a distance, the ``Remote Scientist.'' The Remote Scientist extends laboratory automation capability to the microgravity environment. While the Remote Scientist conceptually encompasses a broad spectrum of elements and functionalities, the development approach taken is to: • establish a baseline capability that is both flexible and versatile • incrementally augment the baseline with additional functions over time. Since last year, the application of the Remote Scientist has changed from protein crystal growth to tissue culture, specifically, the development of skeletal muscle under varying levels of tension. This system includes a series of bioreactor chambers that allow for three-dimensional growth of muscle tissue on a membrane suspended between the two ends of a programmable force transducer that can provide automated or investigator-initiated tension on the developing tissue. A microscope objective mounted on a translation carriage allows for high-resolution microscopy along a large area of the tissue. These images will be mosaiced on orbit to detect features and structures that span multiple images. The use of fluorescence and pseudo-confocal microscopy will maximize the observational capabilities of this system. A series of ground-based experiments have been performed to validate the bioreactor, the force transducer, the translation carriage and the image acquisition capabilities of the Remote Scientist. • The bioreactor is capable of sustaining three dimensional tissue culture growth over time. • The force transducer can be programmed to provide static tension on cells or to simulate either slow or fast growth of underlying tissues in vivo, ranging from 0.2 mm per day to 32 mm per day. • The two-axis translation carriage is capable of scanning the camera along the bioreactor and adjusting the focus with 25 μm resolution. • Time-lapse sequences of images have been acquired, stored and transmitted to a remote computer system. Although the current application of the Remote Scientist technology is the observation and manipulation of a tissue culture growth system, the hardware has been designed to be easily reconfigured to accommodate a multitude of experiments, including animal observation, combustion studies, protein crystal growth, plant growth and aquatic research.

Parameterization of Ca+2-protein interactions for molecular dynamics simulations.

PubMed

Project, Elad; Nachliel, Esther; Gutman, Menachem

2008-05-01

Molecular dynamics simulations of Ca+2 ions near protein were performed with three force fields: GROMOS96, OPLS-AA, and CHARMM22. The simulations reveal major, force-field dependent, inconsistencies in the interaction between the Ca+2 ions with the protein. The variations are attributed to the nonbonded parameterizations of the Ca+2-carboxylates interactions. The simulations results were compared to experimental data, using the Ca+2-HCOO- equilibrium as a model. The OPLS-AA force field grossly overestimates the binding affinity of the Ca+2 ions to the carboxylate whereas the GROMOS96 and CHARMM22 force fields underestimate the stability of the complex. Optimization of the Lennard-Jones parameters for the Ca+2-carboxylate interactions were carried out, yielding new parameters which reproduce experimental data. Copyright 2007 Wiley Periodicals, Inc.

DC biased low-frequency insulating constriction dielectrophoresis for protein biomolecules concentration.

PubMed

Zhang, Peng; Liu, Yuxin

2017-09-01

Sample enrichment or molecules concentration is considered an essential step in sample processing of miniaturized devices aimed at biosensing and bioanalysis. Among all the means involved to achieve this aim, dielectrophoresis (DEP) is increasingly employed in molecules manipulation and concentration because it is non-destructive and high efficiency. This paper presents a methodology to achieve protein concentration utilizing the combination effects of electrokinetics and low frequency insulating dielectrophoresis (iDEP) generated within a microfluidic device, in which a submicron constricted channel was fabricated using DNA molecular combing and replica molding. This fabrication technique avoids using e-beam lithography or other complicated nanochannel fabrication methods, and provides an easy and low cost approach with the flexibility of controlling channel dimensions to create highly constricted channels embedded in a microfluidic device. With theoretical analysis and experiments, we demonstrated that fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) protein molecules can be significantly concentrated to form an arc-shaped band near the constricted channel under the effects of a negative dielectrophoretic force and DC electrokinetic forces within a short period of time. It was also observed that the amplitudes of the applied DC and AC electric fields, the AC frequencies as well as the suspending medium conductivities had strong effects on the concentration responses of the FITC-BSA molecules, including the concentrated area and position, intensities of the focused molecules, and concentration speed. Our method provides a simple and flexible approach for quickly concentrating protein molecules by controlling the applied electric field parameters. The iDEP device reported in this paper can be used as a stand-alone sensor or worked as a pre-concentration module integrated with biosensors for protein biomarker detection. Furthermore, low frequency dielectrophoresis provides practical uses for integrating the concentration module with a portable biosensing system.

Two spatially distinct kinesin-14 proteins, Pkl1 and Klp2, generate collaborative inward forces against kinesin-5 Cut7 in S. pombe.

PubMed

Yukawa, Masashi; Yamada, Yusuke; Yamauchi, Tomoaki; Toda, Takashi

2018-01-04

Kinesin motors play central roles in bipolar spindle assembly. In many eukaryotes, spindle pole separation is driven by kinesin-5, which generates outward force. This outward force is balanced by antagonistic inward force elicited by kinesin-14 and/or dynein. In fission yeast, two kinesin-14 proteins, Pkl1 and Klp2, play an opposing role against the kinesin-5 motor protein Cut7. However, how the two kinesin-14 proteins coordinate individual activities remains elusive. Here, we show that although deletion of either pkl1 or klp2 rescues temperature-sensitive cut7 mutants, deletion of only pkl1 can bypass the lethality caused by cut7 deletion. Pkl1 is tethered to the spindle pole body, whereas Klp2 is localized along the spindle microtubule. Forced targeting of Klp2 to the spindle pole body, however, compensates for Pkl1 functions, indicating that cellular localizations, rather than individual motor specificities, differentiate between the two kinesin-14 proteins. Interestingly, human kinesin-14 (KIFC1 or HSET) can replace either Pkl1 or Klp2. Moreover, overproduction of HSET induces monopolar spindles, reminiscent of the phenotype of Cut7 inactivation. Taken together, this study has uncovered the biological mechanism whereby two different Kinesin-14 motor proteins exert their antagonistic roles against kinesin-5 in a spatially distinct manner. © 2018. Published by The Company of Biologists Ltd.

Preliminary results of human PrPC protein studied by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Nowakowski, Michał; Czapla-Masztafiak, Joanna; Kozak, Maciej; Zhukov, Igor; Zhukova, Lilia; Szlachetko, Jakub; Kwiatek, Wojciech M.

2017-11-01

Neurodegenerative diseases are one of the malfunctions of human nervous system, being a class of complex and prominent pathologies. The human prion Protease Resistant Protein (PrP) is protein regulating copper metabolism in mammalian cells through binding of Cu(II) ions to specific fragments. Nowadays misfolding of this protein is associated with development of prion diseases. Therefore, it is crucial to obtain structural information about coordination of Cu(II) by PrP protein. Herein, we report X-ray absorption spectroscopy (XAS) measurements, carried out on SuperXAS beamline (SLS, PSI Villigen) on PrPC-Cu(II) complexes. Obtained results were compared with theoretical predictions done by FEFF 9.6 software. Complementary to XAS data, Atomic Force Microscopy (AFM) measurements were conducted to obtain low resolution structural information about prepared sample that allow to develop protocol of fixing PrPC molecules on solid substrate used for further experiments. It has been established that folded C-terminal domain of PrPC protein has around 5 nm in diameter. Presented results showed that both XAS and AFM methods are useful tools in detailed examination of complexes of human PrPC either with Cu(II) or with other divalent metal ions.

Assessing the potential of atomistic molecular dynamics simulations to probe reversible protein-protein recognition and binding

PubMed Central

Abriata, Luciano A.; Dal Peraro, Matteo

2015-01-01

Protein-protein recognition and binding are governed by diffusion, noncovalent forces and conformational flexibility, entangled in a way that only molecular dynamics simulations can dissect at high resolution. Here we exploited ubiquitin’s noncovalent dimerization equilibrium to assess the potential of atomistic simulations to reproduce reversible protein-protein binding, by running submicrosecond simulations of systems with multiple copies of the protein at millimolar concentrations. The simulations essentially fail because they lead to aggregates, yet they reproduce some specificity in the binding interfaces as observed in known covalent and noncovalent ubiquitin dimers. Following similar observations in literature we hint at electrostatics and water descriptions as the main liable force field elements, and propose that their optimization should consider observables relevant to multi-protein systems and unfolded proteins. Within limitations, analysis of binding events suggests salient features of protein-protein recognition and binding, to be retested with improved force fields. Among them, that specific configurations of relative direction and orientation seem to trigger fast binding of two molecules, even over 50 Å distances; that conformational selection can take place within surface-to-surface distances of 10 to 40 Å i.e. well before actual intermolecular contact; and that establishment of contacts between molecules further locks their conformations and relative orientations. PMID:26023027

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis.

PubMed

Grinthal, Alison; Adamovic, Ivana; Weiner, Beth; Karplus, Martin; Kleckner, Nancy

2010-02-09

PR65 is the two-layered (alpha-alpha solenoid) HEAT-repeat (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, PI3 kinase target of rapamycin 1) scaffold of protein phosphatase PP2A. Molecular dynamics simulations predict that, at forces expected in living systems, PR65 undergoes (visco-)elastic deformations in response to pulling/pushing on its ends. At lower forces, smooth global flexural and torsional changes occur via even redistribution of stress along the hydrophobic core of the molecule. At intermediate forces, helix-helix separation along one layer ("fracturing") leads to global relaxation plus loss of contact in the other layer to unstack the affected units. Fracture sites are determined by unusual sequences in contiguous interhelix turns. Normal mode analysis of the heterotrimeric PP2A enzyme reveals that its ambient conformational fluctuations are dominated by elastic deformations of PR65, which introduce a mechanical linkage between the separately bound regulatory and catalytic subunits. PR65-dominated fluctuations of PP2A have the effect of opening and closing the enzyme's substrate binding/catalysis interface, as well as altering the positions of certain catalytic residues. These results suggest that substrate binding/catalysis are sensitive to mechanical force. Force could be imposed from the outside (e.g., in PP2A's response to spindle tension) or arise spontaneously (e.g., in PP2A's interaction with unstructured proteins such as Tau, a microtubule-associated Alzheimer's-implicated protein). The presented example supports the view that conformation and function of protein complexes can be modulated by mechanical energy inputs, as well as by chemical energy inputs from ligand binding. Given that helical-repeat proteins are involved in many cellular processes, the findings also encourage the view that mechanical forces may be of widespread importance.

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis

PubMed Central

Grinthal, Alison; Adamovic, Ivana; Weiner, Beth; Karplus, Martin; Kleckner, Nancy

2010-01-01

PR65 is the two-layered (α-α solenoid) HEAT-repeat (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, PI3 kinase target of rapamycin 1) scaffold of protein phosphatase PP2A. Molecular dynamics simulations predict that, at forces expected in living systems, PR65 undergoes (visco-)elastic deformations in response to pulling/pushing on its ends. At lower forces, smooth global flexural and torsional changes occur via even redistribution of stress along the hydrophobic core of the molecule. At intermediate forces, helix–helix separation along one layer (“fracturing”) leads to global relaxation plus loss of contact in the other layer to unstack the affected units. Fracture sites are determined by unusual sequences in contiguous interhelix turns. Normal mode analysis of the heterotrimeric PP2A enzyme reveals that its ambient conformational fluctuations are dominated by elastic deformations of PR65, which introduce a mechanical linkage between the separately bound regulatory and catalytic subunits. PR65-dominated fluctuations of PP2A have the effect of opening and closing the enzyme’s substrate binding/catalysis interface, as well as altering the positions of certain catalytic residues. These results suggest that substrate binding/catalysis are sensitive to mechanical force. Force could be imposed from the outside (e.g., in PP2A’s response to spindle tension) or arise spontaneously (e.g., in PP2A’s interaction with unstructured proteins such as Tau, a microtubule-associated Alzheimer’s-implicated protein). The presented example supports the view that conformation and function of protein complexes can be modulated by mechanical energy inputs, as well as by chemical energy inputs from ligand binding. Given that helical-repeat proteins are involved in many cellular processes, the findings also encourage the view that mechanical forces may be of widespread importance. PMID:20133745

Identifying Floppy and Rigid Regions in Proteins

NASA Astrophysics Data System (ADS)

Jacobs, D. J.; Thorpe, M. F.; Kuhn, L. A.

1998-03-01

In proteins it is possible to separate hard covalent forces involving bond lengths and bond angles from other weak forces. We model the microstructure of the protein as a generic bar-joint truss framework, where the hard covalent forces and strong hydrogen bonds are regarded as rigid bar constraints. We study the mechanical stability of proteins using FIRST (Floppy Inclusions and Rigid Substructure Topography) based on a recently developed combinatorial constraint counting algorithm (the 3D Pebble Game), which is a generalization of the 2D pebble game (D. J. Jacobs and M. F. Thorpe, ``Generic Rigidity: The Pebble Game'', Phys. Rev. Lett.) 75, 4051-4054 (1995) for the special class of bond-bending networks (D. J. Jacobs, "Generic Rigidity in Three Dimensional Bond-bending Networks", Preprint Aug (1997)). This approach is useful in identifying rigid motifs and flexible linkages in proteins, and thereby determines the essential degrees of freedom. We will show some preliminary results from the FIRST analysis on the myohemerythrin and lyozyme proteins.

Assembly and mechanosensory function of focal adhesions: experiments and models.

PubMed

Bershadsky, Alexander D; Ballestrem, Christoph; Carramusa, Letizia; Zilberman, Yuliya; Gilquin, Benoit; Khochbin, Saadi; Alexandrova, Antonina Y; Verkhovsky, Alexander B; Shemesh, Tom; Kozlov, Michael M

2006-04-01

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.

New generation of docking programs: Supercomputer validation of force fields and quantum-chemical methods for docking.

PubMed

Sulimov, Alexey V; Kutov, Danil C; Katkova, Ekaterina V; Ilin, Ivan S; Sulimov, Vladimir B

2017-11-01

Discovery of new inhibitors of the protein associated with a given disease is the initial and most important stage of the whole process of the rational development of new pharmaceutical substances. New inhibitors block the active site of the target protein and the disease is cured. Computer-aided molecular modeling can considerably increase effectiveness of new inhibitors development. Reliable predictions of the target protein inhibition by a small molecule, ligand, is defined by the accuracy of docking programs. Such programs position a ligand in the target protein and estimate the protein-ligand binding energy. Positioning accuracy of modern docking programs is satisfactory. However, the accuracy of binding energy calculations is too low to predict good inhibitors. For effective application of docking programs to new inhibitors development the accuracy of binding energy calculations should be higher than 1kcal/mol. Reasons of limited accuracy of modern docking programs are discussed. One of the most important aspects limiting this accuracy is imperfection of protein-ligand energy calculations. Results of supercomputer validation of several force fields and quantum-chemical methods for docking are presented. The validation was performed by quasi-docking as follows. First, the low energy minima spectra of 16 protein-ligand complexes were found by exhaustive minima search in the MMFF94 force field. Second, energies of the lowest 8192 minima are recalculated with CHARMM force field and PM6-D3H4X and PM7 quantum-chemical methods for each complex. The analysis of minima energies reveals the docking positioning accuracies of the PM7 and PM6-D3H4X quantum-chemical methods and the CHARMM force field are close to one another and they are better than the positioning accuracy of the MMFF94 force field. Copyright © 2017 Elsevier Inc. All rights reserved.

Titin domains progressively unfolded by force are homogenously distributed along the molecule.

PubMed

Bianco, Pasquale; Mártonfalvi, Zsolt; Naftz, Katalin; Kőszegi, Dorina; Kellermayer, Miklós

2015-07-21

Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecule's length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Rate limit of protein elastic response is tether dependent

PubMed Central

Berkovich, Ronen; Hermans, Rodolfo I.; Popa, Ionel; Stirnemann, Guillaume; Garcia-Manyes, Sergi; Berne, Bruce J.; Fernandez, Julio M.

2012-01-01

The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient Deff ∼ 1,200 nm2/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at Deff ∼ 108 nm2/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo with Deff ∼ 104–106 nm2/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk. PMID:22895787

High-frequency electrical stimulation reveals a p38-mTOR signaling module correlated with force-time integral.

PubMed

Rahnert, Jill A; Burkholder, Thomas J

2013-07-15

High-frequency electrical stimulation (HFES) leads to muscle hypertrophy, and attention has been drawn to the high forces involved. However, both mechanical and metabolic stresses occur simultaneously, and both stimuli influence signaling cascades related to protein synthesis. This study aimed to identify the immediate signaling correlates of contraction-induced force and metabolic stresses under the hypothesis that HFES induces growth-related signaling through mechanical stimulation. Force-time integral (FTI) signaling in mouse tibialis anterior muscle was examined by separately manipulating the time of contraction to emphasize the metabolic aspect or the force of contraction to emphasize the mechanical aspect. When FTI was manipulated by changing the total time of activation, phosphorylation of p54 JNK, ERK and p70S6k(T421/S424) was independent of FTI, while phosphorylation of acetyl-CoA carboxylase and p38 correlated with FTI. When FTI was manipulated by changing the force of contraction, p54 JNK, ERK and p70S6k(T421/S424) were again independent of FTI, while phosphorylation of p38 and FAK correlated with FTI. Factor analysis identified a p38-mTOR signaling module that correlated with FTI in both experiments. The consistent link among p38, mTOR and FTI suggests that they form a connected signaling module sensitive to the mechanical aspects of FTI, separate from markers of metabolic load.

Mechanical dynamics in live cells and fluorescence-based force/tension sensors

PubMed Central

Yang, Chao; Zhang, Xiaohan; Guo, Yichen; Meng, Fanjie; Sachs, Frederick; Guo, Jun

2016-01-01

Three signaling systems play the fundamental roles in modulating cell activities: chemical, electrical, and mechanical. While the former two are well studied, the mechanical signaling system is still elusive because of the lack of methods to measure structural forces in real time at cellular and subcellular levels. Indeed, almost all biological processes are responsive to modulation by mechanical forces that trigger dispersive downstream electrical and biochemical pathways. Communication among the three systems is essential to make cells and tissues receptive to environmental changes. Cells have evolved many sophisticated mechanisms for the generation, perception and transduction of mechanical forces, including motor proteins and mechanosensors. In this review, we introduce some background information about mechanical dynamics in live cells, including the ubiquitous mechanical activity, various types of mechanical stimuli exerted on cells and the different mechanosensors. We also summarize recent results obtained using genetically encoded FRET (fluorescence resonance energy transfer)-based force/tension sensors; a new technique used to measure mechanical forces in structural proteins. The sensors have been incorporated into many specific structural proteins and have measured the force gradients in real time within live cells, tissues, and animals. PMID:25958335

High-throughput single-molecule force spectroscopy for membrane proteins

NASA Astrophysics Data System (ADS)

Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

2008-09-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy.

PubMed

Das, Priyadip; Duanias-Assaf, Tal; Reches, Meital

2017-03-06

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Membrane-Mediated Cooperativity of Proteins

NASA Astrophysics Data System (ADS)

Weikl, Thomas R.

2018-04-01

Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.

Protein folding: understanding the role of water and the low Reynolds number environment as the peptide chain emerges from the ribosome and folds.

PubMed

Sen, Siddhartha; Voorheis, H Paul

2014-12-21

The mechanism of protein folding during early stages of the process has three determinants. First, moving water molecules obey the rules of low Reynolds number physics without an inertial component. Molecular movement is instantaneous and size insensitive. Proteins emerging from the ribosome move and rotate without an external force if they change shape, forming and propagating helical structures that increases translocational efficiency. Forward motion ceases when the shape change or propelling force ceases. Second, application of quantum field theory to water structure predicts the spontaneous formation of low density coherent units of fixed size that expel dissolved atmospheric gases. Structured water layers with both coherent and non-coherent domains, form a sheath around the new protein. The surface of exposed hydrophobic amino acids is protected from water contact by small nanobubbles of dissolved atmospheric gases, 5 or 6 molecules on average, that vibrate, attracting even widely separated resonating nanobubbles. This force results from quantum effects, appearing only when the system is within and interacts with an oscillating electromagnetic field. The newly recognized quantum force sharply bends the peptide and is part of a dynamic field determining the pathway of protein folding. Third, the force initiating the tertiary folding of proteins arises from twists at the position of each hydrophobic amino acid, that minimizes surface exposure of the hydrophobic amino acids and propagates along the protein. When the total bend reaches 360°, the leading segment of water sheath intersects the trailing segment. This steric self-intersection expels water from overlapping segments of the sheath and by Newton׳s second law moves the polypeptide chain in an opposite direction. Consequently, with very few exceptions that we enumerate and discuss, tertiary structures are absent from proteins without hydrophobic amino acids, which control the early stages of protein folding and the overall shape of protein. Consequently, proteins only adopt a limited number of forms. The formation of quaternary structures is not necessarily prevented by the absence of hydrophobic amino acids. Copyright © 2014 Elsevier Ltd. All rights reserved.

The dynamics of heat shock system activation in Monomac-6 cells upon Helicobacter pylori infection.

PubMed

Pierzchalski, P; Jastrzebska, M; Link-Lenczowski, P; Leja-Szpak, A; Bonior, J; Jaworek, J; Okon, K; Wojcik, P

2014-12-01

Immune system cells, particularly phagocytes, are exposed to direct contact with pathogens. Because of its nature - elimination of pathogenes - their cytoprotective systems supposed to be quick and forceful. Physiological consequence of phagocytosis for the phagocyte is the apoptotic death to prevent the eventual survival of bacteria as intracellular parasites. However, in some cases, defense systems used by the bacteria force the immune cells to prolong the contact with the pathogen for its effective elimination. Experiments were performed on Monomac-6 cells exposed to live CagA, VacA expressing Helicobacter pylori (H. pylori) over different period of time. Total cellular RNA, cytoplasmic and nuclear proteins were isolated for polymerase chain reaction, Western-blot and electrophoretic mobility shift assay, respectively. We found that Monomac-6 cells infection with H. pylori resulted in the translocation of the entire cellular content of the heat shock protein 70 (HSP70) into the cytoplasm, where its presence could protect cell against toxic products of engulfed bacteria and premature apoptosis. At the same time the nuclear translocation of heat shock factor 1 (HSF-1) and activation of HSP70 gene transcription was noticed. Action of HSP70 might to postpone monocyte apoptosis through protecting cytoplasmic and nuclear proteins from damaging effect of bacterial products, what could be the defending mechanism against the toxic stress caused by engulfed bacteria and provide the immune cell with the sufficient amount of time required for neutralization of the bacteria from phagosomes, even at the expense of temporary lack of the protection of nuclear proteins.

As Simple As Possible, but Not Simpler: Exploring the Fidelity of Coarse-Grained Protein Models for Simulated Force Spectroscopy

PubMed Central

Rottler, Jörg; Plotkin, Steven S.

2016-01-01

Mechanical unfolding of a single domain of loop-truncated superoxide dismutase protein has been simulated via force spectroscopy techniques with both all-atom (AA) models and several coarse-grained models having different levels of resolution: A Gō model containing all heavy atoms in the protein (HA-Gō), the associative memory, water mediated, structure and energy model (AWSEM) which has 3 interaction sites per amino acid, and a Gō model containing only one interaction site per amino acid at the Cα position (Cα-Gō). To systematically compare results across models, the scales of time, energy, and force had to be suitably renormalized in each model. Surprisingly, the HA-Gō model gives the softest protein, exhibiting much smaller force peaks than all other models after the above renormalization. Clustering to render a structural taxonomy as the protein unfolds showed that the AA, HA-Gō, and Cα-Gō models exhibit a single pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches only after the protein is about half-unfolded. The AWSEM model shows a single dominant unfolding pathway over the whole range of unfolding, in contrast to all other models. TM alignment, clustering analysis, and native contact maps show that the AWSEM pathway has however the most structural similarity to the AA model at high nativeness, but the least structural similarity to the AA model at low nativeness. In comparison to the AA model, the sequence of native contact breakage is best predicted by the HA-Gō model. All models consistently predict a similar unfolding mechanism for early force-induced unfolding events, but diverge in their predictions for late stage unfolding events when the protein is more significantly disordered. PMID:27898663

As Simple As Possible, but Not Simpler: Exploring the Fidelity of Coarse-Grained Protein Models for Simulated Force Spectroscopy.

PubMed

Habibi, Mona; Rottler, Jörg; Plotkin, Steven S

2016-11-01

Mechanical unfolding of a single domain of loop-truncated superoxide dismutase protein has been simulated via force spectroscopy techniques with both all-atom (AA) models and several coarse-grained models having different levels of resolution: A Gō model containing all heavy atoms in the protein (HA-Gō), the associative memory, water mediated, structure and energy model (AWSEM) which has 3 interaction sites per amino acid, and a Gō model containing only one interaction site per amino acid at the Cα position (Cα-Gō). To systematically compare results across models, the scales of time, energy, and force had to be suitably renormalized in each model. Surprisingly, the HA-Gō model gives the softest protein, exhibiting much smaller force peaks than all other models after the above renormalization. Clustering to render a structural taxonomy as the protein unfolds showed that the AA, HA-Gō, and Cα-Gō models exhibit a single pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches only after the protein is about half-unfolded. The AWSEM model shows a single dominant unfolding pathway over the whole range of unfolding, in contrast to all other models. TM alignment, clustering analysis, and native contact maps show that the AWSEM pathway has however the most structural similarity to the AA model at high nativeness, but the least structural similarity to the AA model at low nativeness. In comparison to the AA model, the sequence of native contact breakage is best predicted by the HA-Gō model. All models consistently predict a similar unfolding mechanism for early force-induced unfolding events, but diverge in their predictions for late stage unfolding events when the protein is more significantly disordered.

Hydration water dynamics and instigation of protein structuralrelaxation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russo, Daniela; Hura, Greg; Head-Gordon, Teresa

2003-09-01

Until a critical hydration level is reached, proteins do not function. This critical level of hydration is analogous to a similar lack of protein function observed for temperatures below a dynamical temperature range of 180-220K that also is connected to the dynamics of protein surface water. Restoration of some enzymatic activity is observed in partially hydrated protein powders, sometimes corresponding to less than a single hydration layer on the protein surface, which indicates that the dynamical and structural properties of the surface water is intimately connected to protein stability and function. Many elegant studies using both experiment and simulation havemore » contributed important information about protein hydration structure and timescales. The molecular mechanism of the solvent motion that is required to instigate the protein structural relaxation above a critical hydration level or transition temperature has yet to be determined. In this work we use experimental quasi-elastic neutron scattering (QENS) and molecular dynamics simulation to investigate hydration water dynamics near a greatly simplified protein system. We consider the hydration water dynamics near the completely deuterated N-acetyl-leucine-methylamide (NALMA) solute, a hydrophobic amino acid side chain attached to a polar blocked polypeptide backbone, as a function of concentration between 0.5M-2.0M under ambient conditions. We note that roughly 50-60% of a folded protein's surface is equally distributed between hydrophobic and hydrophilic domains, domains whose lengths are on the order of a few water diameters, that justify our study of hydration dynamics of this simple model protein system. The QENS experiment was performed at the NIST Center for Neutron Research, using the disk chopper time of flight spectrometer (DCS). In order to separate the translational and rotational components in the spectra, two sets of experiments were carried out using different incident neutron wavelengths of 7.5{angstrom} and 5.5{angstrom} to give two different time resolutions. All the spectra have been measure at room temperature. The spectra were corrected for the sample holder contribution and normalized using the vanadium standard. The resulting data were analyzed with DAVE programs (http://www.ncnr.nist.gov/dave/). The AMBER force field and SPCE water model were used for modeling the NALMA solute and water, respectively. For the analysis of the water dynamics in the NALMA aqueous solutions, we performed simulations of a dispersed solute configuration consistent with our previous structural analysis, where we had primarily focused on the structural organization of these peptide solutions and their connection to protein folding. Further details of the QENS experiment and molecular dynamics simulations are reported elsewhere.« less

Electrostatic forces govern the binding mechanism of intrinsically disordered histone chaperones

PubMed Central

Liu, Chuanbo; Wang, Tianshu; Bai, Yawen; Wang, Jin

2017-01-01

A unified picture to understand the protein recognition and function must include the native binding complex structure ensembles and the underlying binding mechanisms involved in specific biological processes. However, quantifications of both binding complex structures and dynamical mechanisms are still challenging for IDP. In this study, we have investigated the underlying molecular mechanism of the chaperone Chz1 and histone H2A.Z-H2B association by equilibrium and kinetic stopped-flow fluorescence spectroscopy. The dependence of free energy and kinetic rate constant on electrolyte mean activity coefficient and urea concentration are uncovered. Our results indicate a previous unseen binding kinetic intermediate. An initial conformation selection step of Chz1 is also revealed before the formation of this intermediate state. Based on these observations, a mixed mechanism of three steps including both conformation selection and induced fit is proposed. By combination of the ion- and denaturant-induced experiments, we demonstrate that electrostatic forces play a dominant role in the recognition of bipolar charged intrinsically disordered protein Chz1 to its preferred partner H2A.Z-H2B. Both the intra-chain and inter-chain electrostatic interactions have direct impacts on the native collapsed structure and binding mechanism. PMID:28552960

Recent applications of boxed molecular dynamics: a simple multiscale technique for atomistic simulations.

PubMed

Booth, Jonathan; Vazquez, Saulo; Martinez-Nunez, Emilio; Marks, Alison; Rodgers, Jeff; Glowacki, David R; Shalashilin, Dmitrii V

2014-08-06

In this paper, we briefly review the boxed molecular dynamics (BXD) method which allows analysis of thermodynamics and kinetics in complicated molecular systems. BXD is a multiscale technique, in which thermodynamics and long-time dynamics are recovered from a set of short-time simulations. In this paper, we review previous applications of BXD to peptide cyclization, solution phase organic reaction dynamics and desorption of ions from self-assembled monolayers (SAMs). We also report preliminary results of simulations of diamond etching mechanisms and protein unfolding in atomic force microscopy experiments. The latter demonstrate a correlation between the protein's structural motifs and its potential of mean force. Simulations of these processes by standard molecular dynamics (MD) is typically not possible, because the experimental time scales are very long. However, BXD yields well-converged and physically meaningful results. Compared with other methods of accelerated MD, our BXD approach is very simple; it is easy to implement, and it provides an integrated approach for simultaneously obtaining both thermodynamics and kinetics. It also provides a strategy for obtaining statistically meaningful dynamical results in regions of configuration space that standard MD approaches would visit only very rarely.

Guided by curvature: shaping cells by coupling curved membrane proteins and cytoskeletal forces.

PubMed

Gov, N S

2018-05-26

Eukaryote cells have flexible membranes that allow them to have a variety of dynamical shapes. The shapes of the cells serve important biological functions, both for cells within an intact tissue, and during embryogenesis and cellular motility. How cells control their shapes and the structures that they form on their surface has been a subject of intensive biological research, exposing the building blocks that cells use to deform their membranes. These processes have also drawn the interest of theoretical physicists, aiming to develop models based on physics, chemistry and nonlinear dynamics. Such models explore quantitatively different possible mechanisms that the cells can employ to initiate the spontaneous formation of shapes and patterns on their membranes. We review here theoretical work where one such class of mechanisms was investigated: the coupling between curved membrane proteins, and the cytoskeletal forces that they recruit. Theory indicates that this coupling gives rise to a rich variety of membrane shapes and dynamics, while experiments indicate that this mechanism appears to drive many cellular shape changes.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

Mechanics of torque generation in the bacterial flagellar motor

PubMed Central

Mandadapu, Kranthi K.; Nirody, Jasmine A.; Berry, Richard M.; Oster, George

2015-01-01

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual “power stroke.” Specifically, we propose that ion-induced conformational changes about a proline “hinge” residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque–speed and speed–ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator. PMID:26216959

Simplified TiO2 force fields for studies of its interaction with biomolecules

NASA Astrophysics Data System (ADS)

Luan, Binquan; Huynh, Tien; Zhou, Ruhong

2015-06-01

Engineered TiO2 nanoparticles have been routinely applied in nanotechnology, as well as in cosmetics and food industries. Despite active experimental studies intended to clarify TiO2's biological effects, including potential toxicity, the relation between experimentally inferred nanotoxicity and industry standards for safely applying nanoparticles remains somewhat ambiguous with justified concerns. Supplemental to experiments, molecular dynamics simulations have proven to be efficacious in investigating the molecular mechanism of a biological process occurring at nanoscale. In this article, to facilitate the nanotoxicity and nanomedicine research related to this important metal oxide, we provide a simplified force field, based on the original Matsui-Akaogi force field but compatible to the Lennard-Jones potentials normally used in modeling biomolecules, for simulating TiO2 nanoparticles interacting with biomolecules. The force field parameters were tested in simulating the bulk structure of TiO2, TiO2 nanoparticle-water interaction, as well as the adsorption of proteins on the TiO2 nanoparticle. We demonstrate that these simulation results are consistent with experimental data/observations. We expect that simulations will help to better understand the interaction between TiO2 and molecules.

Mechanics of torque generation in the bacterial flagellar motor.

PubMed

Mandadapu, Kranthi K; Nirody, Jasmine A; Berry, Richard M; Oster, George

2015-08-11

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual "power stroke." Specifically, we propose that ion-induced conformational changes about a proline "hinge" residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque-speed and speed-ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

Protein crystal growth

NASA Technical Reports Server (NTRS)

2001-01-01

Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

Molecular Mechanics

PubMed Central

Vanommeslaeghe, Kenno; Guvench, Olgun; MacKerell, Alexander D.

2014-01-01

Molecular Mechanics (MM) force fields are the methods of choice for protein simulations, which are essential in the study of conformational flexibility. Given the importance of protein flexibility in drug binding, MM is involved in most if not all Computational Structure-Based Drug Discovery (CSBDD) projects. This section introduces the reader to the fundamentals of MM, with a special emphasis on how the target data used in the parametrization of force fields determine their strengths and weaknesses. Variations and recent developments such as polarizable force fields are discussed. The section ends with a brief overview of common force fields in CSBDD. PMID:23947650

Protein crystals as scanned probes for recognition atomic force microscopy.

PubMed

Wickremasinghe, Nissanka S; Hafner, Jason H

2005-12-01

Lysozyme crystal growth has been localized at the tip of a conventional silicon nitride cantilever through seeded nucleation. After cross-linking with glutaraldehyde, lysozyme protein crystal tips image gold nanoparticles and grating standards with a resolution comparable to that of conventional tips. Force spectra between the lysozyme crystal tips and surfaces covered with antilysozyme reveal an adhesion force that drops significantly upon blocking with free lysozyme, thus confirming that lysozyme crystal tips can detect molecular recognition interactions.

Update of the ATTRACT force field for the prediction of protein-protein binding affinity.

PubMed

Chéron, Jean-Baptiste; Zacharias, Martin; Antonczak, Serge; Fiorucci, Sébastien

2017-06-05

Determining the protein-protein interactions is still a major challenge for molecular biology. Docking protocols has come of age in predicting the structure of macromolecular complexes. However, they still lack accuracy to estimate the binding affinities, the thermodynamic quantity that drives the formation of a complex. Here, an updated version of the protein-protein ATTRACT force field aiming at predicting experimental binding affinities is reported. It has been designed on a dataset of 218 protein-protein complexes. The correlation between the experimental and predicted affinities reaches 0.6, outperforming most of the available protocols. Focusing on a subset of rigid and flexible complexes, the performance raises to 0.76 and 0.69, respectively. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Solvent Effects on Protein Folding/Unfolding

NASA Astrophysics Data System (ADS)

García, A. E.; Hillson, N.; Onuchic, J. N.

Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.

Amino acid analogues bind to carbon nanotube via π-π interactions: Comparison of molecular mechanical and quantum mechanical calculations

NASA Astrophysics Data System (ADS)

Yang, Zaixing; Wang, Zhigang; Tian, Xingling; Xiu, Peng; Zhou, Ruhong

2012-01-01

Understanding the interaction between carbon nanotubes (CNTs) and biomolecules is essential to the CNT-based nanotechnology and biotechnology. Some recent experiments have suggested that the π-π stacking interactions between protein's aromatic residues and CNTs might play a key role in their binding, which raises interest in large scale modeling of protein-CNT complexes and associated π-π interactions at atomic detail. However, there is concern on the accuracy of classical fixed-charge molecular force fields due to their classical treatments and lack of polarizability. Here, we study the binding of three aromatic residue analogues (mimicking phenylalanine, tyrosine, and tryptophan) and benzene to a single-walled CNT, and compare the molecular mechanical (MM) calculations using three popular fixed-charge force fields (OPLSAA, AMBER, and CHARMM), with quantum mechanical (QM) calculations using the density-functional tight-binding method with the inclusion of dispersion correction (DFTB-D). Two typical configurations commonly found in π-π interactions are used, one with the aromatic rings parallel to the CNT surface (flat), and the other perpendicular (edge). Our calculations reveal that compared to the QM results the MM approaches can appropriately reproduce the strength of π-π interactions for both configurations, and more importantly, the energy difference between them, indicating that the various contributions to π-π interactions have been implicitly included in the van der Waals parameters of the standard MM force fields. Meanwhile, these MM models are less accurate in predicting the exact structural binding patterns (matching surface), meaning there are still rooms to be improved. In addition, we have provided a comprehensive and reliable QM picture for the π-π interactions of aromatic molecules with CNTs in gas phase, which might be used as a benchmark for future force field developments.

Amino acid analogues bind to carbon nanotube via π-π interactions: comparison of molecular mechanical and quantum mechanical calculations.

PubMed

Yang, Zaixing; Wang, Zhigang; Tian, Xingling; Xiu, Peng; Zhou, Ruhong

2012-01-14

Understanding the interaction between carbon nanotubes (CNTs) and biomolecules is essential to the CNT-based nanotechnology and biotechnology. Some recent experiments have suggested that the π-π stacking interactions between protein's aromatic residues and CNTs might play a key role in their binding, which raises interest in large scale modeling of protein-CNT complexes and associated π-π interactions at atomic detail. However, there is concern on the accuracy of classical fixed-charge molecular force fields due to their classical treatments and lack of polarizability. Here, we study the binding of three aromatic residue analogues (mimicking phenylalanine, tyrosine, and tryptophan) and benzene to a single-walled CNT, and compare the molecular mechanical (MM) calculations using three popular fixed-charge force fields (OPLSAA, AMBER, and CHARMM), with quantum mechanical (QM) calculations using the density-functional tight-binding method with the inclusion of dispersion correction (DFTB-D). Two typical configurations commonly found in π-π interactions are used, one with the aromatic rings parallel to the CNT surface (flat), and the other perpendicular (edge). Our calculations reveal that compared to the QM results the MM approaches can appropriately reproduce the strength of π-π interactions for both configurations, and more importantly, the energy difference between them, indicating that the various contributions to π-π interactions have been implicitly included in the van der Waals parameters of the standard MM force fields. Meanwhile, these MM models are less accurate in predicting the exact structural binding patterns (matching surface), meaning there are still rooms to be improved. In addition, we have provided a comprehensive and reliable QM picture for the π-π interactions of aromatic molecules with CNTs in gas phase, which might be used as a benchmark for future force field developments.

Targeted Changes of the Cell Wall Proteome Influence Candida albicans Ability to Form Single- and Multi-strain Biofilms

PubMed Central

Walker, Louise A.; Martin-Yken, Hélène; Dague, Etienne; Legrand, Mélanie; Lee, Keunsook; Chauvel, Murielle; Firon, Arnaud; Rossignol, Tristan; Richard, Mathias L.; Munro, Carol A.; Bachellier-Bassi, Sophie; d'Enfert, Christophe

2014-01-01

Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (∼10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence. PMID:25502890

Alchemical prediction of hydration free energies for SAMPL

PubMed Central

Mobley, David L.; Liu, Shaui; Cerutti, David S.; Swope, William C.; Rice, Julia E.

2013-01-01

Hydration free energy calculations have become important tests of force fields. Alchemical free energy calculations based on molecular dynamics simulations provide a rigorous way to calculate these free energies for a particular force field, given sufficient sampling. Here, we report results of alchemical hydration free energy calculations for the set of small molecules comprising the 2011 Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) challenge. Our calculations are largely based on the Generalized Amber Force Field (GAFF) with several different charge models, and we achieved RMS errors in the 1.4-2.2 kcal/mol range depending on charge model, marginally higher than what we typically observed in previous studies1-5. The test set consists of ethane, biphenyl, and a dibenzyl dioxin, as well as a series of chlorinated derivatives of each. We found that, for this set, using high-quality partial charges from MP2/cc-PVTZ SCRF RESP fits provided marginally improved agreement with experiment over using AM1-BCC partial charges as we have more typically done, in keeping with our recent findings5. Switching to OPLS Lennard-Jones parameters with AM1-BCC charges also improves agreement with experiment. We also find a number of chemical trends within each molecular series which we can explain, but there are also some surprises, including some that are captured by the calculations and some that are not. PMID:22198475

Membrane Disruption Mechanism of a Prion Peptide (106-126) Investigated by Atomic Force Microscopy, Raman and Electron Paramagnetic Resonance Spectroscopy.

PubMed

Pan, Jianjun; Sahoo, Prasana K; Dalzini, Annalisa; Hayati, Zahra; Aryal, Chinta M; Teng, Peng; Cai, Jianfeng; Rodriguez Gutierrez, Humberto; Song, Likai

2017-05-18

A fragment of the human prion protein spanning residues 106-126 (PrP106-126) recapitulates many essential properties of the disease-causing protein such as amyloidogenicity and cytotoxicity. PrP106-126 has an amphipathic characteristic that resembles many antimicrobial peptides (AMPs). Therefore, the toxic effect of PrP106-126 could arise from a direct association of monomeric peptides with the membrane matrix. Several experimental approaches are employed to scrutinize the impacts of monomeric PrP106-126 on model lipid membranes. Porous defects in planar bilayers are observed by using solution atomic force microscopy. Adding cholesterol does not impede defect formation. A force spectroscopy experiment shows that PrP106-126 reduces Young's modulus of planar lipid bilayers. We use Raman microspectroscopy to study the effect of PrP106-126 on lipid atomic vibrational dynamics. For phosphatidylcholine lipids, PrP106-126 disorders the intrachain conformation, while the interchain interaction is not altered; for phosphatidylethanolamine lipids, PrP106-126 increases the interchain interaction, while the intrachain conformational order remains similar. We explain the observed differences by considering different modes of peptide insertion. Finally, electron paramagnetic resonance spectroscopy shows that PrP106-126 progressively decreases the orientational order of lipid acyl chains in magnetically aligned bicelles. Together, our experimental data support the proposition that monomeric PrP106-126 can disrupt lipid membranes by using similar mechanisms found in AMPs.

Membrane Disruption Mechanism of a Prion Peptide (106-126) Investigated by Atomic Force Microscopy, Raman and Electron Paramagnetic Resonance Spectroscopy

PubMed Central

Pan, Jianjun; Sahoo, Prasana K.; Dalzini, Annalisa; Hayati, Zahra; Aryal, Chinta M.; Teng, Peng; Cai, Jianfeng; Gutierrez, Humberto Rodriguez; Song, Likai

2018-01-01

A fragment of the human prion protein spanning residues 106-126 (PrP106-126) recapitulates many essential properties of the disease-causing protein such as amyloidogenicity and cytotoxicity. PrP106-126 has an amphipathic characteristic that resembles many antimicrobial peptides (AMPs). Therefore, the toxic effect of PrP106-126 could arise from a direct association of monomeric peptides with membrane matrix. Several experimental approaches are employed to scrutinize the impacts of monomeric PrP106-126 on model lipid membranes. Porous defects in planar bilayers are observed by using solution atomic force microscopy. Adding cholesterol does not impede defect formation. Force spectroscopy experiment shows that PrP106-126 reduces Young’s modulus of planar lipid bilayers. We use Raman microspectroscopy to study the effect of PrP106-126 on lipid vibrational dynamics. For phosphatidylcholine lipids, PrP106-126 disorders the intra-chain conformation, while the inter-chain interaction is not altered; for phosphatidylethanolamine lipids, PrP106-126 increases the inter-chain interaction, while the intra-chain conformational order remains similar. We explain the observed differences by considering different modes of peptide insertion. Finally, electron paramagnetic resonance spectroscopy shows that PrP106-126 progressively decreases the orientational order of lipid acyl chains in magnetically aligned bicelles. Together, our experimental data support the proposition that monomeric PrP106-126 can disrupt lipid membranes by using similar mechanisms found in AMPs. PMID:28459565

Mapping flexible protein domains at subnanometer resolution with the atomic force microscope.

PubMed

Müller, D J; Fotiadis, D; Engel, A

1998-06-23

The mapping of flexible protein domains with the atomic force microscope is reviewed. Examples discussed are the bacteriorhodopsin from Halobacterium salinarum, the head-tail-connector from phage phi29, and the hexagonally packed intermediate layer from Deinococcus radiodurans which all were recorded in physiological buffer solution. All three proteins undergo reversible structural changes that are reflected in standard deviation maps calculated from aligned topographs of individual protein complexes. Depending on the lateral resolution (up to 0.8 nm) flexible surface regions can ultimately be correlated with individual polypeptide loops. In addition, multivariate statistical classification revealed the major conformations of the protein surface.

Scaling and self-organized criticality in proteins II

PubMed Central

Phillips, J. C.

2009-01-01

The complexity of proteins is substantially simplified by regarding them as archetypical examples of self-organized criticality (SOC). To test this idea and to elaborate it, this article applies the Moret–Zebende (MZ) SOC hydrophobicity scale to transport repeat proteins of the HEAT superfamily, importin β, and transportin, as well as the export protein Cse1p, and their ubiquitous cargo manager Ran. The difference between the MZ scale and conventional hydrophobicity scales reflects long-range conformational forces that are central to protein functionality. These compete with long-range Coulomb forces associated with cationic and anionic side chains in a revealing way. PMID:19124778

Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

PubMed Central

MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

2008-01-01

Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

Lipid and protein composition as driving force for multiple sclerosis

NASA Astrophysics Data System (ADS)

Beck, Roy; Shaharabani, Rona

Physical models and experiments often reduce the number of components aiming to address the fundamental mechanisms. Nevertheless, the inherent heterogeneity is an essential ingredient in the biological context. We present our recent efforts to model and understand the development of multiple sclerosis (MS) from a biophysical perspective. Myelin sheath is a multilamellar complex of various lipids and proteins that surround axons and acts as an insulating layer for proper nerve conduction. In MS the myelin structure is disrupted impairing its function. Previous studies showed that MS is correlated with small lipid composition variation and reduction in the adhesive myelin basic protein. We found that such alterations result in pathological phase transition from a lamellar to inverted hexagonal that involve enhanced local curvature. Similar curvatures are also found in vivo in diseased myelin sheaths. Since the etiology and recovery pathways of MS are currently unclear, these findings delineate novel functional roles to dominant constituents in cytoplasmic myelin sheaths, shed new light on mechanisms disrupting lipid-protein complexes, and suggest new courses for diagnosis and treatment for MS.

Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy

PubMed Central

2016-01-01

The function of bioenergetic membranes is strongly influenced by the spatial arrangement of their constituent membrane proteins. Atomic force microscopy (AFM) can be used to probe protein organization at high resolution, allowing individual proteins to be identified. However, previous AFM studies of biological membranes have typically required that curved membranes are ruptured and flattened during sample preparation, with the possibility of disruption of the native protein arrangement or loss of proteins. Imaging native, curved membranes requires minimal tip–sample interaction in both lateral and vertical directions. Here, long-range tip–sample interactions are reduced by optimizing the imaging buffer. Tapping mode AFM with high-resonance-frequency small and soft cantilevers, in combination with a high-speed AFM, reduces the forces due to feedback error and enables application of an average imaging force of tens of piconewtons. Using this approach, we have imaged the membrane organization of intact vesicular bacterial photosynthetic “organelles”, chromatophores. Despite the highly curved nature of the chromatophore membrane and lack of direct support, the resolution was sufficient to identify the photosystem complexes and quantify their arrangement in the native state. Successive imaging showed the proteins remain surprisingly static, with minimal rotation or translation over several-minute time scales. High-order assemblies of RC-LH1-PufX complexes are observed, and intact ATPases are successfully imaged. The methods developed here are likely to be applicable to a broad range of protein-rich vesicles or curved membrane systems, which are an almost ubiquitous feature of native organelles. PMID:28114766

Synergistic interactions of lipids and myelin basic protein

NASA Astrophysics Data System (ADS)

Hu, Yufang; Doudevski, Ivo; Wood, Denise; Moscarello, Mario; Husted, Cynthia; Genain, Claude; Zasadzinski, Joseph A.; Israelachvili, Jacob

2004-09-01

This report describes force measurements and atomic force microscope imaging of lipid-protein interactions that determine the structure of a model membrane system that closely mimics the myelin sheath. Our results suggest that noncovalent, mainly electrostatic and hydrophobic, interactions are responsible for the multilamellar structure and stability of myelin. We find that myelin basic protein acts as a lipid coupler between two apposed bilayers and as a lipid "hole-filler," effectively preventing defect holes from developing. From our protein-mediated-adhesion and force-distance measurements, we develop a simple quantitative model that gives a reasonably accurate picture of the molecular mechanism and adhesion of bilayer-bridging proteins by means of noncovalent interactions. The results and model indicate that optimum myelin adhesion and stability depend on the difference between, rather than the product of, the opposite charges on the lipid bilayers and myelin basic protein, as well as on the repulsive forces associated with membrane fluidity, and that small changes in any of these parameters away from the synergistically optimum values can lead to large changes in the adhesion or even its total elimination. Our results also show that the often-asked question of which membrane species, the lipids or the proteins, are the "important ones" may be misplaced. Both components work synergistically to provide the adhesion and overall structure. A better appreciation of the mechanism of this synergy may allow for a better understanding of stacked and especially myelin membrane structures and may lead to better treatments for demyelinating diseases such as multiple sclerosis. lipid-protein interactions | myelin membrane structure | membrane adhesion | membrane regeneration/healing | demyelinating diseases

Self diffusion of interacting membrane proteins.

PubMed Central

Abney, J R; Scalettar, B A; Owicki, J C

1989-01-01

A two-dimensional version of the generalized Smoluchowski equation is used to analyze the time (or distance) dependent self diffusion of interacting membrane proteins in concentrated membrane systems. This equation provides a well established starting point for descriptions of the diffusion of particles that interact through both direct and hydrodynamic forces; in this initial work only the effects of direct interactions are explicitly considered. Data describing diffusion in the presence of hard-core repulsions, soft repulsions, and soft repulsions with weak attractions are presented. The effect that interactions have on the self-diffusion coefficient of a real protein molecule from mouse liver gap junctions is also calculated. The results indicate that self diffusion is always inhibited by direct interactions; this observation is interpreted in terms of the caging that will exist at finite protein concentration. It is also noted that, over small distance scales, the diffusion coefficient is determined entirely by the very strong Brownian forces; therefore, as a function of displacement the self-diffusion coefficient decays (rapidly) from its value at infinite dilution to its steady-state interaction-averaged value. The steady-state self-diffusion coefficient describes motion over distance scales that range from approximately 10 nm to cellular dimensions and is the quantity measured in fluorescence recovery after photobleaching experiments. The short-ranged behavior of the diffusion coefficient is important on the interparticle-distance scale and may therefore influence the rate at which nearest-neighbor collisional processes take place. The hard-disk theoretical results presented here are in excellent agreement with lattice Monte-Carlo results obtained by other workers. The concentration dependence of experimentally measured diffusion coefficients of antibody-hapten complexes bound to the membrane surface is consistent with that predicted by the theory. The variation in experimental diffusion coefficients of integral membrane proteins is greater than that predicted by the theory, and may also reflect protein-induced perturbations in membrane viscosity. PMID:2720077

Inhibitors of SOD1 Interaction as an Approach to Slow the Progressive Spread of ALS Symptoms

DTIC Science & Technology

2016-07-01

luciferase enzyme can be split into 2 halves. These 2 halves can be forced to reconstitute an active enzyme if they are brought together by some...force. In our assay, this force is the normal interaction that occurs when 2 individual SOD1 proteins come together to form a normal active enzyme ...Using recombinant DNA, we create fusion proteins of SOD1 and each half of the luciferase enzyme . In the past year, we have characterized and optimized

An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export.

PubMed

Freudl, R; Schwarz, H; Stierhof, Y D; Gamon, K; Hindennach, I; Henning, U

1986-08-25

Pulse-chase experiments were performed to follow the export of the Escherichia coli outer membrane protein OmpA. Besides the pro-OmpA protein, which carries a 21-residue signal sequence, three species of ompA gene products were distinguishable. One probably represented an incomplete nascent chain, another the mature protein in the outer membrane, and the third, designated imp-OmpA (immature processed), a protein which was already processed but apparently was still associated with the plasma membrane. The pro- and imp-OmpA proteins could be characterized more fully by using a strain overproducing the ompA gene products; pro- and imp-OmpA accumulated in large amounts. It could be shown that the imp- and pro-OmpA proteins differ markedly in conformation from the OmpA protein. The imp-OmpA, but not the pro-OmpA, underwent a conformational change and gained phage receptor activity upon addition of lipopolysaccharide. Utilizing a difference in detergent solubility between the two polypeptides and employing immunoelectron microscopy, it could be demonstrated that the pro-OmpA protein accumulated in the cytoplasm while the imp-OmpA was present in the periplasmic space. The results suggest that the pro-OmpA protein, bound to the plasma membrane, is processed, and the resulting imp-OmpA, still associated with the plasma membrane, recognizes the lipid A moiety of the lipopolysaccharide. The resulting conformational change may then force the protein into the outer membrane.

Environmental Assessment for Air Force Research Laboratory Space Vehicles Integrated Experiments Division Office Space at Kirtland Air Force Base, Albuquerque, New Mexico

DTIC Science & Technology

2005-06-01

AIR FORCE RESEARCH LABORATORY SPACE VEHICLES INTEGRATED EXPERMENTS DIVISION OFFICE SPACE AT KIRTLAND AIR FORCE ... Kirtland Air Force Base (KAFB). The office building would house the Air Force Research Laboratory Space Vehicles Integrated Experiments Division...ADDRESS(ES) Air Force Research Laboratory ,Space Vehicles Directorate,3550 Aberdeen Ave. SE, Kirtland

Effective harmonic approach to helix proteins

NASA Astrophysics Data System (ADS)

Choi, Sung Nam Peter

Recent laser-induced experiments have shown that on the time scale of long range tertiary contact formation, the kinetics of the helix-coil interconversion are very rapid (1). The notion that secondary structures form first before long range tertiary contacts are made, suggests that the helix-coil interconversion is one key in understanding the protein folding problem. We developed a method of generating an alpha-helix conformation of any residue using AMBER 4.0, the Protein Data Bank (PDB), and a series of pattern matching translation and rotation operations. We have chosen to study Alanine for its highly non polar nature, and Glutamine for its highly polar characteristic. Using the information obtained from the above method, analysis of vibrational lattice dynamics are carried out on polymers Poly-alpha-L alanine (PLA) and Poly-alpha-L glutamic acid (PLGA). The lattice dynamics method called Modified Self- consistent Harmonic Approach theory (MSHA) was used in carrying out the analysis of the PLA and PLGA helix-coil transition. MSHA was originally developed for the DNA molecule by Prohofsky et al. In this model, the molecule is considered as a one dimensional, repeating unit cell of an infinite helix. The lattice was modeled with harmonic force constants. Anharmonicity of hydrogen bond force constants at different temperatures are introduced along with the effects of water on the polar PLGA and the non polar PLA. Using MSHA, we calculated the opening bond probabilities of PLGA at different temperatures. The experimentally observed helix to coil relaxation time τ* of PLGA was inversely compared to our results with a correlation coefficient of 0.8424 (5, 13, 58). These experiments were done at temperatures ranging from 295 K to 310 K. Our calculation also yielded critical melting temperatures of Tc=317 K for PLGA and Tc=347 K for PLA. The calculated acoustic compressional velocities were 4.78 km/s and 4.84 km/s for PLA and PLGA respectively. Our calculation clearly indicates that the MSHA methodology, along with the use of AMBER 4.0, yields a good physical model of helix to coil transition of proteins.

Dynamics of living matter: can we ``see'' collective motions in proteins?

NASA Astrophysics Data System (ADS)

Hekstra, Doeke

2015-03-01

Proteins are ideal model systems for quantitative study of the interplay of physical and evolutionary forces. Collective, anharmonic motions of amino acid residues within proteins are thought to be central to their function, and to explain, in large part, the complex dependence of protein function on its constituent parts. Currently, the experimental characterization of such motions poses a major stumbling block on the way to a physical understanding of protein function and evolution. We are addressing this problem in two ways. First, alternate conformations of protein residues can often be distinguished in the electron density estimated from room-temperature X-ray crystallography. The dense packing of residues in the folded protein requires that such conformational variations must propagate through networks of amino acids to preclude local steric clashes. Fraser and colleagues showed that such steric conflicts can be used to extract contact networks of residues collectively switching conformation. We ask if these networks are conserved over homologous sequences and connected to the functional reaction coordinate, both of which would demonstrate their fundamental importance. I will describe initial results for the family of PDZ domains: small ligand-binding proteins for which a network of energetically and conformationally coupled residues controlling ligand affinity has been demonstrated previously by a range of methods. Second, the analysis of collective motions in proteins, by nearly any means, is indirect: nothing is seen moving. To directly induce and ``see'' motions on a range of time scales, we developed a new approach based on (a) electric field pulses to induce motions within a protein crystal and (b) time-resolved crystallography to observe these motions. Since proteins generically have a heterogeneous, modifiable charge distribution, this method could provide a powerful, general way of probing the collective motions, and excited states, of proteins in kinetic and atomic detail. I will present initial experiments showing the method is feasible. Taken together, these experiments begin to provide a basis for the development of a physical theory of proteins consistent with their function and adaptation - the source of their survival throughout the evolutionary process.

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer’s disease

PubMed Central

Grégoire, Catherine; Marco, Sergio; Thimonier, Jean; Duplan, Laure; Laurine, Emmanuelle; Chauvin, Jean-Paul; Michel, Bernard; Peyrot, Vincent; Verdier, Jean-Michel

2001-01-01

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre-clinical stages of Alzheimer’s disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimer’s disease. After self-proteolysis, the N-terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three-dimensional X-ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple-helical filament (QHF-litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils. PMID:11432819

Predictive energy landscapes for folding membrane protein assemblies

NASA Astrophysics Data System (ADS)

Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

2015-12-01

We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

Learning about Biomolecular Solvation from Water in Protein Crystals.

PubMed

Altan, Irem; Fusco, Diana; Afonine, Pavel V; Charbonneau, Patrick

2018-03-08

Water occupies typically 50% of a protein crystal and thus significantly contributes to the diffraction signal in crystallography experiments. Separating its contribution from that of the protein is, however, challenging because most water molecules are not localized and are thus difficult to assign to specific density peaks. The intricateness of the protein-water interface compounds this difficulty. This information has, therefore, not often been used to study biomolecular solvation. Here, we develop a methodology to surmount in part this difficulty. More specifically, we compare the solvent structure obtained from diffraction data for which experimental phasing is available to that obtained from constrained molecular dynamics (MD) simulations. The resulting spatial density maps show that commonly used MD water models are only partially successful at reproducing the structural features of biomolecular solvation. The radial distribution of water is captured with only slightly higher accuracy than its angular distribution, and only a fraction of the water molecules assigned with high reliability to the crystal structure is recovered. These differences are likely due to shortcomings of both the water models and the protein force fields. Despite these limitations, we manage to infer protonation states of some of the side chains utilizing MD-derived densities.

Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks

NASA Astrophysics Data System (ADS)

Bayburt, Timothy H.; Sligar, Stephen G.

2002-05-01

The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.

Ultrastable cellulosome-adhesion complex tightens under load.

PubMed

Schoeler, Constantin; Malinowska, Klara H; Bernardi, Rafael C; Milles, Lukas F; Jobst, Markus A; Durner, Ellis; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Schulten, Klaus; Gaub, Hermann E; Nash, Michael A

2014-12-08

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand-receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600-750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass.

Application of the maximum entropy principle to determine ensembles of intrinsically disordered proteins from residual dipolar couplings.

PubMed

Sanchez-Martinez, M; Crehuet, R

2014-12-21

We present a method based on the maximum entropy principle that can re-weight an ensemble of protein structures based on data from residual dipolar couplings (RDCs). The RDCs of intrinsically disordered proteins (IDPs) provide information on the secondary structure elements present in an ensemble; however even two sets of RDCs are not enough to fully determine the distribution of conformations, and the force field used to generate the structures has a pervasive influence on the refined ensemble. Two physics-based coarse-grained force fields, Profasi and Campari, are able to predict the secondary structure elements present in an IDP, but even after including the RDC data, the re-weighted ensembles differ between both force fields. Thus the spread of IDP ensembles highlights the need for better force fields. We distribute our algorithm in an open-source Python code.

PIEZO channel protein naturally expressed in human breast cancer cell MDA-MB-231 as probed by atomic force microscopy

NASA Astrophysics Data System (ADS)

Weng, Yuanqi; Yan, Fei; Chen, Runkang; Qian, Ming; Ou, Yun; Xie, Shuhong; Zheng, Hairong; Li, Jiangyu

2018-05-01

Mechanical stimuli drives many physiological processes through mechanically activated channels, and the recent discovery of PIEZO channel has generated great interests in its mechanotransduction. Many previous researches investigated PIEZO proteins by transcribing them in cells that originally have no response to mechanical stimulation, or by forming PIEZO-combined complexes in vitro, and few studied PIEZO protein's natural characteristics in cells. In this study we show that MDA-MB-231, a malignant cell in human breast cancer cell line, expresses the mechanosensitive behavior of PIEZO in nature without extra treatment, and we report its characteristics in response to localized mechanical stimulation under an atomic force microscope, wherein a correlation between the force magnitude applied and the channel opening probability is observed. The results on PIEZO of MDA-MB-231 can help establish a basis of preventing and controlling of human breast cancer cell via mechanical forces.

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore.

PubMed

Tang, Jilin; Krajcikova, Daniela; Zhu, Rong; Ebner, Andreas; Cutting, Simon; Gruber, Hermann J; Barak, Imrich; Hinterdorfer, Peter

2007-01-01

Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores. Copyright (c) 2007 John Wiley & Sons, Ltd.

Parallel Force Assay for Protein-Protein Interactions

PubMed Central

Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

Parallel force assay for protein-protein interactions.

PubMed

Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

PubMed

Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J

2003-10-01

Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

Monitoring developmental force distributions in reconstituted embryonic epithelia.

PubMed

Przybyla, L; Lakins, J N; Sunyer, R; Trepat, X; Weaver, V M

2016-02-01

The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell-ECM and cell-cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization. Copyright © 2015 Elsevier Inc. All rights reserved.

Deposition kinetics of extracellular polymeric substances (EPS) on silica in monovalent and divalent salts.

PubMed

Zhu, Pingting; Long, Guoyu; Ni, Jinren; Tong, Meiping

2009-08-01

The deposition kinetics of extracellular polymeric substances (EPS) on silica surfaces were examined in both monovalent and divalent solutions under a variety of environmentally relevant ionic strength and pH conditions by employing a quartz crystal microbalance with dissipation (DCM-D). Soluble EPS (SEPS) and bound EPS (BEPS) were extracted from four bacterial strains with different characteristics. Maximum favorable deposition rates (k(fa)) were observed for all EPS at low ionic strengths in both NaCl and CaCl2 solutions. With the increase of ionic strength, k(fa) decreased due to the simultaneous occurrence of EPS aggregation in solutions. Deposition efficiency (alpha; the ratio of deposition rates obtained under unfavorable versus corresponding favorable conditions) for all EPS increased with increasing ionic strength in both NaCl and CaCl2 solutions, which agreed with the trends of zeta potentials and was consistent with the classic Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Comparison of alpha for SEPS and BEPS extracted from the same strain showed that the trends of alpha did not totally agree with trends of zeta potentials, indicating the deposition kinetics of EPS on silica surfaces were not only controlled by DLVO interactions, but also non-DLVO forces. Close comparison of alpha for EPS extracted from different sources showed alpha increased with increasing proteins to polysaccharides ratio. Subsequent experiments for EPS extracted from the same strain but with different proteins to polysaccharides ratios and from activated sludge also showed that alpha were largest for EPS with greatest proteins to polysaccharides ratio. Additional experiments for pure protein and solutions with different pure proteins to pure saccharides ratios further corroborated that larger proteins to polysaccharides ratio resulted in greater EPS deposition.

On artifacts in single-molecule force spectroscopy

PubMed Central

Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

2015-01-01

In typical force spectroscopy experiments, a small biomolecule is attached to a soft polymer linker that is pulled with a relatively large bead or cantilever. At constant force, the total extension stochastically changes between two (or more) values, indicating that the biomolecule undergoes transitions between two (or several) conformational states. In this paper, we consider the influence of the dynamics of the linker and mesoscopic pulling device on the force-dependent rate of the conformational transition extracted from the time dependence of the total extension, and the distribution of rupture forces in force-clamp and force-ramp experiments, respectively. For these different experiments, we derive analytic expressions for the observables that account for the mechanical response and dynamics of the pulling device and linker. Possible artifacts arise when the characteristic times of the pulling device and linker become comparable to, or slower than, the lifetimes of the metastable conformational states, and when the highly anharmonic regime of stretched linkers is probed at high forces. We also revisit the problem of relating force-clamp and force-ramp experiments, and identify a linker and loading rate-dependent correction to the rates extracted from the latter. The theory provides a framework for both the design and the quantitative analysis of force spectroscopy experiments by highlighting, and correcting for, factors that complicate their interpretation. PMID:26540730

Nonlinear finite-element analysis of nanoindentation of viral capsids

NASA Astrophysics Data System (ADS)

Gibbons, Melissa M.; Klug, William S.

2007-03-01

Recent atomic force microscope (AFM) nanoindentation experiments measuring mechanical response of the protein shells of viruses have provided a quantitative description of their strength and elasticity. To better understand and interpret these measurements, and to elucidate the underlying mechanisms, this paper adopts a course-grained modeling approach within the framework of three-dimensional nonlinear continuum elasticity. Homogeneous, isotropic, elastic, thick-shell models are proposed for two capsids: the spherical cowpea chlorotic mottle virus (CCMV), and the ellipsocylindrical bacteriophage ϕ29 . As analyzed by the finite-element method, these models enable parametric characterization of the effects of AFM tip geometry, capsid dimensions, and capsid constitutive descriptions. The generally nonlinear force response of capsids to indentation is shown to be insensitive to constitutive particulars, and greatly influenced by geometric and kinematic details. Nonlinear stiffening and softening of the force response is dependent on the AFM tip dimensions and shell thickness. Fits of the models capture the roughly linear behavior observed in experimental measurements and result in estimates of Young’s moduli of ≈280-360MPa for CCMV and ≈4.5GPa for ϕ29 .

Evaluation of solvation free energies for small molecules with the AMOEBA polarizable force field

PubMed Central

Mohamed, Noor Asidah; Bradshaw, Richard T.

2016-01-01

The effects of electronic polarization in biomolecular interactions will differ depending on the local dielectric constant of the environment, such as in solvent, DNA, proteins, and membranes. Here the performance of the AMOEBA polarizable force field is evaluated under nonaqueous conditions by calculating the solvation free energies of small molecules in four common organic solvents. Results are compared with experimental data and equivalent simulations performed with the GAFF pairwise‐additive force field. Although AMOEBA results give mean errors close to “chemical accuracy,” GAFF performs surprisingly well, with statistically significantly more accurate results than AMOEBA in some solvents. However, for both models, free energies calculated in chloroform show worst agreement to experiment and individual solutes are consistently poor performers, suggesting non‐potential‐specific errors also contribute to inaccuracy. Scope for the improvement of both potentials remains limited by the lack of high quality experimental data across multiple solvents, particularly those of high dielectric constant. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. PMID:27757978

A closer look at the complex hydrophilic/hydrophobic interactions forces at the human hair surface

NASA Astrophysics Data System (ADS)

Baghdadli, N.; Luengo, G. S.; Recherche, L.

2008-03-01

The complex chemical structure of the hair surface is far from being completely understood. Current understanding is based on Rivett's model1 that was proposed to explain the macroscopic hydrophobic nature of the surface of natural hair. In this model covalently-linked fatty acids are chemically grafted to the amorphous protein (keratin) through a thio-ester linkage2,3. Nevertheless, experience like wetting and electrical properties of human hair surface4 shows that the complexity of the hair surface is not fully understand based on this model in literature. Recent studies in our laboratory show for the first time microscopic evidence of the heterogeneous physico-chemical character of the hair surface. By using Chemical Force Microscopy, the presence of hydrophobic and ionic species are detected and localized, before and after a cosmetic treatment (bleaching). Based on force curve analysis the mapping of the local distribution of hydrophilic and hydrophobic groups of hair surface is obtained. A discussion on a more plausible hair model and its implications will be presented based on these new results.

Force, Torque and Stiffness: Interactions in Perceptual Discrimination

PubMed Central

Wu, Bing; Klatzky, Roberta L.; Hollis, Ralph L.

2011-01-01

Three experiments investigated whether force and torque cues interact in haptic discrimination of force, torque and stiffness, and if so, how. The statistical relation between force and torque was manipulated across four experimental conditions: Either one type of cue varied while the other was constant, or both varied so as to be positively correlated, negatively correlated, or uncorrelated. Experiment 1 showed that the subjects’ ability to discriminate force was improved by positively correlated torque but impaired with uncorrelated torque, as compared to the constant torque condition. Corresponding effects were found in Experiment 2 for the influence of force on torque discrimination. These findings indicate that force and torque are integrated in perception, rather than being processed as separate dimensions. A further experiment demonstrated facilitation of stiffness discrimination by correlated force and torque, whether the correlation was positive or negative. The findings suggest new means of augmenting haptic feedback to facilitate perception of the properties of soft objects. PMID:21359137

Forces Driving Chaperone Action

PubMed Central

Koldewey, Philipp; Stull, Frederick; Horowitz, Scott; Martin, Raoul; Bardwell, James C. A.

2016-01-01

SUMMARY It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client’s affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins. PMID:27293188

Atomic force microscopy and spectroscopy to probe single membrane proteins in lipid bilayers.

PubMed

Sapra, K Tanuj

2013-01-01

The atomic force microscope (AFM) has opened vast avenues hitherto inaccessible to the biological scientist. The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever stylus is aptly termed as a "lab on a tip" owing to its versatility as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples assert that the AFM can be used to study the mechanical properties and monitor processes of single proteins and single cells, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of membrane proteins. Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theoretical, and practical skills.

Multidrug Transport Protein NorM from Vibrio cholerae Simultaneously Couples to Sodium- and Proton-Motive Force*

PubMed Central

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W.

2014-01-01

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na+ or H+. We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na+ or H+ binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. PMID:24711447

Soft Vibrational Modes Predict Breaking Events during Force-Induced Protein Unfolding.

PubMed

Habibi, Mona; Plotkin, Steven S; Rottler, Jörg

2018-02-06

We investigate the correlation between soft vibrational modes and unfolding events in simulated force spectroscopy of proteins. Unfolding trajectories are obtained from molecular dynamics simulations of a Gō model of a monomer of a mutant of superoxide dismutase 1 protein containing all heavy atoms in the protein, and a normal mode analysis is performed based on the anisotropic network model. We show that a softness map constructed from the superposition of the amplitudes of localized soft modes correlates with unfolding events at different stages of the unfolding process. Soft residues are up to eight times more likely to undergo disruption of native structure than the average amino acid. The memory of the softness map is retained for extensions of up to several nanometers, but decorrelates more rapidly during force drops. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Local traction force in the proximal leading process triggers nuclear translocation during neuronal migration.

PubMed

Umeshima, Hiroki; Nomura, Ken-Ichi; Yoshikawa, Shuhei; Hörning, Marcel; Tanaka, Motomu; Sakuma, Shinya; Arai, Fumihito; Kaneko, Makoto; Kengaku, Mineko

2018-04-05

Somal translocation in long bipolar neurons is regulated by actomyosin contractile forces, yet the precise spatiotemporal sites of force generation are unknown. Here we investigate the force dynamics generated during somal translocation using traction force microscopy. Neurons with a short leading process generated a traction force in the growth cone and counteracting forces in the leading and trailing processes. In contrast, neurons with a long leading process generated a force dipole with opposing traction forces in the proximal leading process during nuclear translocation. Transient accumulation of actin filaments was observed at the dipole center of the two opposing forces, which was abolished by inhibition of myosin II activity. A swelling in the leading process emerged and generated a traction force that pulled the nucleus when nuclear translocation was physically hampered. The traction force in the leading process swelling was uncoupled from somal translocation in neurons expressing a dominant negative mutant of the KASH protein, which disrupts the interaction between cytoskeletal components and the nuclear envelope. Our results suggest that the leading process is the site of generation of actomyosin-dependent traction force in long bipolar neurons, and that the traction force is transmitted to the nucleus via KASH proteins. Copyright © 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.

Further along the Road Less Traveled: AMBER ff15ipq, an Original Protein Force Field Built on a Self-Consistent Physical Model

PubMed Central

2016-01-01

We present the AMBER ff15ipq force field for proteins, the second-generation force field developed using the Implicitly Polarized Q (IPolQ) scheme for deriving implicitly polarized atomic charges in the presence of explicit solvent. The ff15ipq force field is a complete rederivation including more than 300 unique atomic charges, 900 unique torsion terms, 60 new angle parameters, and new atomic radii for polar hydrogens. The atomic charges were derived in the context of the SPC/Eb water model, which yields more-accurate rotational diffusion of proteins and enables direct calculation of nuclear magnetic resonance (NMR) relaxation parameters from molecular dynamics simulations. The atomic radii improve the accuracy of modeling salt bridge interactions relative to contemporary fixed-charge force fields, rectifying a limitation of ff14ipq that resulted from its use of pair-specific Lennard-Jones radii. In addition, ff15ipq reproduces penta-alanine J-coupling constants exceptionally well, gives reasonable agreement with NMR relaxation rates, and maintains the expected conformational propensities of structured proteins/peptides, as well as disordered peptides—all on the microsecond (μs) time scale, which is a critical regime for drug design applications. These encouraging results demonstrate the power and robustness of our automated methods for deriving new force fields. All parameters described here and the mdgx program used to fit them are included in the AmberTools16 distribution. PMID:27399642

Silk from crickets: a new twist on spinning.

PubMed

Walker, Andrew A; Weisman, Sarah; Church, Jeffrey S; Merritt, David J; Mudie, Stephen T; Sutherland, Tara D

2012-01-01

Raspy crickets (Orthoptera: Gryllacrididae) are unique among the orthopterans in producing silk, which is used to build shelters. This work studied the material composition and the fabrication of cricket silk for the first time. We examined silk-webs produced in captivity, which comprised cylindrical fibers and flat films. Spectra obtained from micro-Raman experiments indicated that the silk is composed of protein, primarily in a beta-sheet conformation, and that fibers and films are almost identical in terms of amino acid composition and secondary structure. The primary sequences of four silk proteins were identified through a mass spectrometry/cDNA library approach. The most abundant silk protein was large in size (300 and 220 kDa variants), rich in alanine, glycine and serine, and contained repetitive sequence motifs; these are features which are shared with several known beta-sheet forming silk proteins. Convergent evolution at the molecular level contrasts with development by crickets of a novel mechanism for silk fabrication. After secretion of cricket silk proteins by the labial glands they are fabricated into mature silk by the labium-hypopharynx, which is modified to allow the controlled formation of either fibers or films. Protein folding into beta-sheet structure during silk fabrication is not driven by shear forces, as is reported for other silks.

A Maximum-Likelihood Approach to Force-Field Calibration.

PubMed

Zaborowski, Bartłomiej; Jagieła, Dawid; Czaplewski, Cezary; Hałabis, Anna; Lewandowska, Agnieszka; Żmudzińska, Wioletta; Ołdziej, Stanisław; Karczyńska, Agnieszka; Omieczynski, Christian; Wirecki, Tomasz; Liwo, Adam

2015-09-28

A new approach to the calibration of the force fields is proposed, in which the force-field parameters are obtained by maximum-likelihood fitting of the calculated conformational ensembles to the experimental ensembles of training system(s). The maximum-likelihood function is composed of logarithms of the Boltzmann probabilities of the experimental conformations, calculated with the current energy function. Because the theoretical distribution is given in the form of the simulated conformations only, the contributions from all of the simulated conformations, with Gaussian weights in the distances from a given experimental conformation, are added to give the contribution to the target function from this conformation. In contrast to earlier methods for force-field calibration, the approach does not suffer from the arbitrariness of dividing the decoy set into native-like and non-native structures; however, if such a division is made instead of using Gaussian weights, application of the maximum-likelihood method results in the well-known energy-gap maximization. The computational procedure consists of cycles of decoy generation and maximum-likelihood-function optimization, which are iterated until convergence is reached. The method was tested with Gaussian distributions and then applied to the physics-based coarse-grained UNRES force field for proteins. The NMR structures of the tryptophan cage, a small α-helical protein, determined at three temperatures (T = 280, 305, and 313 K) by Hałabis et al. ( J. Phys. Chem. B 2012 , 116 , 6898 - 6907 ), were used. Multiplexed replica-exchange molecular dynamics was used to generate the decoys. The iterative procedure exhibited steady convergence. Three variants of optimization were tried: optimization of the energy-term weights alone and use of the experimental ensemble of the folded protein only at T = 280 K (run 1); optimization of the energy-term weights and use of experimental ensembles at all three temperatures (run 2); and optimization of the energy-term weights and the coefficients of the torsional and multibody energy terms and use of experimental ensembles at all three temperatures (run 3). The force fields were subsequently tested with a set of 14 α-helical and two α + β proteins. Optimization run 1 resulted in better agreement with the experimental ensemble at T = 280 K compared with optimization run 2 and in comparable performance on the test set but poorer agreement of the calculated folding temperature with the experimental folding temperature. Optimization run 3 resulted in the best fit of the calculated ensembles to the experimental ones for the tryptophan cage but in much poorer performance on the training set, suggesting that use of a small α-helical protein for extensive force-field calibration resulted in overfitting of the data for this protein at the expense of transferability. The optimized force field resulting from run 2 was found to fold 13 of the 14 tested α-helical proteins and one small α + β protein with the correct topologies; the average structures of 10 of them were predicted with accuracies of about 5 Å C(α) root-mean-square deviation or better. Test simulations with an additional set of 12 α-helical proteins demonstrated that this force field performed better on α-helical proteins than the previous parametrizations of UNRES. The proposed approach is applicable to any problem of maximum-likelihood parameter estimation when the contributions to the maximum-likelihood function cannot be evaluated at the experimental points and the dimension of the configurational space is too high to construct histograms of the experimental distributions.

In silico folding of a three helix protein and characterization of its free-energy landscape in an all-atom force field.

PubMed

Herges, T; Wenzel, W

2005-01-14

We report the reproducible first-principles folding of the 40 amino-acid, three-helix headpiece of the HIV accessory protein in a recently developed all-atom free-energy force field. Six of 20 simulations using an adapted basin-hopping method converged to better than 3 A backbone rms deviation to the experimental structure. Using over 60 000 low-energy conformations of this protein, we constructed a decoy tree that completely characterizes its folding funnel.

In Silico Folding of a Three Helix Protein and Characterization of Its Free-Energy Landscape in an All-Atom Force Field

NASA Astrophysics Data System (ADS)

Herges, T.; Wenzel, W.

2005-01-01

We report the reproducible first-principles folding of the 40 amino-acid, three-helix headpiece of the HIV accessory protein in a recently developed all-atom free-energy force field. Six of 20 simulations using an adapted basin-hopping method converged to better than 3Å backbone rms deviation to the experimental structure. Using over 60 000 low-energy conformations of this protein, we constructed a decoy tree that completely characterizes its folding funnel.

Atomic Force Microscopy for Protein Detection and Their Physicoсhemical Characterization

PubMed Central

Bukharina, Natalia S.; Archakov, Alexander I.; Ivanov, Yuri D.

2018-01-01

This review is focused on the atomic force microscopy (AFM) capabilities to study the properties of protein biomolecules and to detect the proteins in solution. The possibilities of application of a wide range of measuring techniques and modes for visualization of proteins, determination of their stoichiometric characteristics and physicochemical properties, are analyzed. Particular attention is paid to the use of AFM as a molecular detector for detection of proteins in solutions at low concentrations, and also for determination of functional properties of single biomolecules, including the activity of individual molecules of enzymes. Prospects for the development of AFM in combination with other methods for studying biomacromolecules are discussed. PMID:29642632

Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects.

PubMed

Batthyány, Carlos; Bartesaghi, Silvina; Mastrogiovanni, Mauricio; Lima, Analía; Demicheli, Verónica; Radi, Rafael

2017-03-01

"Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function. Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation. The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.

The nature of the force-induced conformation transition of dsDNA studied by using single molecule force spectroscopy.

PubMed

Liu, Ningning; Bu, Tianjia; Song, Yu; Zhang, Wei; Li, Jinjing; Zhang, Wenke; Shen, Jiacong; Li, Hongbin

2010-06-15

Single-stranded DNA binding proteins (SSB) interact with single-stranded DNA (ssDNA) specifically. Taking advantage of this character, we have employed Bacillus subtilis SSB protein to investigate the nature of force-induced conformation transition of double-stranded DNA (dsDNA) by using AFM-based single molecule force spectroscopy (SMFS) technique. Our results show that, when a dsDNA is stretched beyond its contour length, the dsDNA is partially melted, producing some ssDNA segments which can be captured by SSB proteins. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. Furthermore, the effect of proflavine, a DNA intercalator, on the SSB-DNA interactions has been investigated, and the results indicate that the proflavine-saturated dsDNA can be stabilized to the extent that the dsDNA will no longer melt into ssDNA under the mechanical force even up to 150 pN, and no SSB-DNA interactions are detectable.

Tackling force-field bias in protein folding simulations: folding of Villin HP35 and Pin WW domains in explicit water.

PubMed

Mittal, Jeetain; Best, Robert B

2010-08-04

The ability to fold proteins on a computer has highlighted the fact that existing force fields tend to be biased toward a particular type of secondary structure. Consequently, force fields for folding simulations are often chosen according to the native structure, implying that they are not truly "transferable." Here we show that, while the AMBER ff03 potential is known to favor helical structures, a simple correction to the backbone potential (ff03( *)) results in an unbiased energy function. We take as examples the 35-residue alpha-helical Villin HP35 and 37 residue beta-sheet Pin WW domains, which had not previously been folded with the same force field. Starting from unfolded configurations, simulations of both proteins in Amber ff03( *) in explicit solvent fold to within 2.0 A RMSD of the experimental structures. This demonstrates that a simple backbone correction results in a more transferable force field, an important requirement if simulations are to be used to interpret folding mechanism. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex.

PubMed

Arsenovic, Paul T; Bathula, Kranthidhar; Conway, Daniel E

2017-04-11

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a Förster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A new insight into diffusional escape from a biased cylindrical trap

NASA Astrophysics Data System (ADS)

Berezhkovskii, Alexander M.; Dagdug, Leonardo; Bezrukov, Sergey M.

2017-09-01

Recent experiments with single biological nanopores, as well as single-molecule fluorescence spectroscopy and pulling studies of protein and nucleic acid folding raised a number of questions that stimulated theoretical and computational investigations of barrier crossing dynamics. The present paper addresses a closely related problem focusing on trajectories of Brownian particles that escape from a cylindrical trap in the presence of a force F parallel to the cylinder axis. To gain new insights into the escape dynamics, we analyze the "fine structure" of these trajectories. Specifically, we divide trajectories into two segments: a looping segment, when a particle unsuccessfully tries to escape returning to the trap bottom again and again, and a direct-transit segment, when it finally escapes moving without touching the bottom. Analytical expressions are derived for the Laplace transforms of the probability densities of the durations of the two segments. These expressions are used to find the mean looping and direct-transit times as functions of the biasing force F. It turns out that the force-dependences of the two mean times are qualitatively different. The mean looping time monotonically increases as F decreases, approaching exponential F-dependence at large negative forces pushing the particle towards the trap bottom. In contrast to this intuitively appealing behavior, the mean direct-transit time shows rather counterintuitive behavior: it decreases as the force magnitude, |F|, increases independently of whether the force pushes the particles to the trap bottom or to the exit from the trap, having a maximum at F = 0.

Focal Contacts as Mechanosensors

PubMed Central

Riveline, Daniel; Zamir, Eli; Balaban, Nathalie Q.; Schwarz, Ulrich S.; Ishizaki, Toshimasa; Narumiya, Shuh; Kam, Zvi; Geiger, Benjamin; Bershadsky, Alexander D.

2001-01-01

The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force. PMID:11402062

Defense Horizons. Number 25, March 2003. Biology and the Battlefield

DTIC Science & Technology

2003-03-01

Joint Task Force is notified that they will deploy to Southwest Asia within 36 hours. The team will insert 65 miles behind enemy forces, in the...10 Ibid. 11 Carol Ezzell , “Proteins Rule,” Scientific American 286, no. 4 (April 2002), 40–47. 12 “Protein,” accessed at <http://encarta.msn.com/encnet

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

NASA Astrophysics Data System (ADS)

Casuso, Ignacio; Khao, Jonathan; Chami, Mohamed; Paul-Gilloteaux, Perrine; Husain, Mohamed; Duneau, Jean-Pierre; Stahlberg, Henning; Sturgis, James N.; Scheuring, Simon

2012-08-01

For cells to function properly, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.

Hydrogen bonds are a primary driving force for de novo protein folding

DOE PAGES

Lee, Schuyler; Wang, Chao; Liu, Haolin; ...

2017-11-10

The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1–153; AID 153 ), an optimized in vitro folding procedure was derived to obtain large amounts of AID 153 , which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the proteinmore » after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis -to- trans proline isomerization, or vice versa , during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo . It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.« less

Hydrogen bonds are a primary driving force for de novo protein folding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Schuyler; Wang, Chao; Liu, Haolin

The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1–153; AID 153 ), an optimized in vitro folding procedure was derived to obtain large amounts of AID 153 , which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the proteinmore » after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis -to- trans proline isomerization, or vice versa , during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo . It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.« less

Interfacial layers from the protein HFBII hydrophobin: dynamic surface tension, dilatational elasticity and relaxation times.

PubMed

Alexandrov, Nikola A; Marinova, Krastanka G; Gurkov, Theodor D; Danov, Krassimir D; Kralchevsky, Peter A; Stoyanov, Simeon D; Blijdenstein, Theodorus B J; Arnaudov, Luben N; Pelan, Eddie G; Lips, Alex

2012-06-15

The pendant-drop method (with drop-shape analysis) and Langmuir trough are applied to investigate the characteristic relaxation times and elasticity of interfacial layers from the protein HFBII hydrophobin. Such layers undergo a transition from fluid to elastic solid films. The transition is detected as an increase in the error of the fit of the pendant-drop profile by means of the Laplace equation of capillarity. The relaxation of surface tension after interfacial expansion follows an exponential-decay law, which indicates adsorption kinetics under barrier control. The experimental data for the relaxation time suggest that the adsorption rate is determined by the balance of two opposing factors: (i) the barrier to detachment of protein molecules from bulk aggregates and (ii) the attraction of the detached molecules by the adsorption layer due to the hydrophobic surface force. The hydrophobic attraction can explain why a greater surface coverage leads to a faster adsorption. The relaxation of surface tension after interfacial compression follows a different, square-root law. Such behavior can be attributed to surface diffusion of adsorbed protein molecules that are condensing at the periphery of interfacial protein aggregates. The surface dilatational elasticity, E, is determined in experiments on quick expansion or compression of the interfacial protein layers. At lower surface pressures (<11 mN/m) the experiments on expansion, compression and oscillations give close values of E that are increasing with the rise of surface pressure. At higher surface pressures, E exhibits the opposite tendency and the data are scattered. The latter behavior can be explained with a two-dimensional condensation of adsorbed protein molecules at the higher surface pressures. The results could be important for the understanding and control of dynamic processes in foams and emulsions stabilized by hydrophobins, as well as for the modification of solid surfaces by adsorption of such proteins. Copyright © 2012 Elsevier Inc. All rights reserved.

Impact of short range hydrophobic interactions and long range electrostatic forces on the aggregation kinetics of a monoclonal antibody and a dual-variable domain immunoglobulin at low and high concentrations.

PubMed

Kumar, Vineet; Dixit, Nitin; Zhou, Liqiang Lisa; Fraunhofer, Wolfgang

2011-12-12

The purpose of this work was to determine the nature of long and short-range forces governing protein aggregation kinetics at low and high concentrations for a monoclonal antibody (IgG1) and a dual-variable-domain immunoglobulin (DVD-Ig). Protein-protein interactions (PPI) were studied under dilute conditions by utilizing the methods of static (B(22)) and dynamic light scattering (k(D)). PPI in solutions containing minimal ionic strengths were characterized to get detailed insights into the impact of ionic strength on aggregation. Microcalorimetry and susceptibility to denature at air-liquid interface were used to assess the tertiary structure and quiescent stability studies were conducted to study aggregation characteristics. Results for IgG1 showed that electrostatic interactions governed protein aggregation kinetics both under dilute and concentrated conditions (i.e., 5 mg/mL and 150 mg/mL). For DVD-Ig molecules, on the other hand, although electrostatic interactions governed protein aggregation under dilute conditions, hydrophobic forces clearly determined the kinetics at high concentrations. This manuscript shows for the first time that short-range hydrophobic interactions can outweigh electrostatic forces and play an important role in determining protein aggregation at high concentrations. Additionally, results show that although higher-order virial coefficients become significant under low ionic strength conditions, removal of added charges may be used to enhance the aggregation stability of dilute protein formulations. Copyright © 2011 Elsevier B.V. All rights reserved.

Formation of a cylindrical bridge in cell division

NASA Astrophysics Data System (ADS)

Citron, Daniel; Schmidt, Laura E.; Reichl, Elizabeth; Ren, Yixin; Robinson, Douglas; Zhang, Wendy W.

2007-11-01

In nature, the shape transition associated with the division of a mother cell into two daughter cells proceeds via a variety of routes. In the cylinder-thinning route, which has been observed in Dictyostelium and most animal cells, the mother cell first forms a broad bridge-like region, also known as a furrow, between two daughter cells. The furrow then rapidly evolves into a cylindrical bridge, which thins and eventually severs the mother cell into two. The fundamental mechanism underlying this division route is not understood. Recent experiments on Dictyostelium found that, while the cylinder-thinning route persists even when key actin cross-linking proteins are missing, it is disrupted by the removal of force-generating myosin-II proteins. Other measurements revealed that mutant cells lacking myosin-II have a much more uniform tension over the cell surface than wild-type cells. This suggests that tension variation may be important. Here we use a fluid model, previously shown to reproduce the thinning dynamics [Zhang & Robinson, PNAS 102, 7186 (2005)], to test this idea. Consistent with the experiments, the model shows that the cylinder formation process occurs regardless of the exact viscoelastic properties of the cell. In contrast to the experiments, a tension variation in the model hinders, rather then expedites, the cylinder formation.

Micromechanics of human mitotic chromosomes

NASA Astrophysics Data System (ADS)

Sun, Mingxuan; Kawamura, Ryo; Marko, John F.

2011-02-01

Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

PubMed Central

Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P.

2016-01-01

Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

Are Current Atomistic Force Fields Accurate Enough to Study Proteins in Crowded Environments?

PubMed Central

Petrov, Drazen; Zagrovic, Bojan

2014-01-01

The high concentration of macromolecules in the crowded cellular interior influences different thermodynamic and kinetic properties of proteins, including their structural stabilities, intermolecular binding affinities and enzymatic rates. Moreover, various structural biology methods, such as NMR or different spectroscopies, typically involve samples with relatively high protein concentration. Due to large sampling requirements, however, the accuracy of classical molecular dynamics (MD) simulations in capturing protein behavior at high concentration still remains largely untested. Here, we use explicit-solvent MD simulations and a total of 6.4 µs of simulated time to study wild-type (folded) and oxidatively damaged (unfolded) forms of villin headpiece at 6 mM and 9.2 mM protein concentration. We first perform an exhaustive set of simulations with multiple protein molecules in the simulation box using GROMOS 45a3 and 54a7 force fields together with different types of electrostatics treatment and solution ionic strengths. Surprisingly, the two villin headpiece variants exhibit similar aggregation behavior, despite the fact that their estimated aggregation propensities markedly differ. Importantly, regardless of the simulation protocol applied, wild-type villin headpiece consistently aggregates even under conditions at which it is experimentally known to be soluble. We demonstrate that aggregation is accompanied by a large decrease in the total potential energy, with not only hydrophobic, but also polar residues and backbone contributing substantially. The same effect is directly observed for two other major atomistic force fields (AMBER99SB-ILDN and CHARMM22-CMAP) as well as indirectly shown for additional two (AMBER94, OPLS-AAL), and is possibly due to a general overestimation of the potential energy of protein-protein interactions at the expense of water-water and water-protein interactions. Overall, our results suggest that current MD force fields may distort the picture of protein behavior in biologically relevant crowded environments. PMID:24854339

Effects of 3D virtual haptics force feedback on brand personality perception: the mediating role of physical presence in advergames.

PubMed

Jin, Seung-A Annie

2010-06-01

This study gauged the effects of force feedback in the Novint Falcon haptics system on the sensory and cognitive dimensions of a virtual test-driving experience. First, in order to explore the effects of tactile stimuli with force feedback on users' sensory experience, feelings of physical presence (the extent to which virtual physical objects are experienced as actual physical objects) were measured after participants used the haptics interface. Second, to evaluate the effects of force feedback on the cognitive dimension of consumers' virtual experience, this study investigated brand personality perception. The experiment utilized the Novint Falcon haptics controller to induce immersive virtual test-driving through tactile stimuli. The author designed a two-group (haptics stimuli with force feedback versus no force feedback) comparison experiment (N = 238) by manipulating the level of force feedback. Users in the force feedback condition were exposed to tactile stimuli involving various force feedback effects (e.g., terrain effects, acceleration, and lateral forces) while test-driving a rally car. In contrast, users in the control condition test-drove the rally car using the Novint Falcon but were not given any force feedback. Results of ANOVAs indicated that (a) users exposed to force feedback felt stronger physical presence than those in the no force feedback condition, and (b) users exposed to haptics stimuli with force feedback perceived the brand personality of the car to be more rugged than those in the control condition. Managerial implications of the study for product trial in the business world are discussed.

Mechanical Force-induced TGFB1 Increases Expression of SOST/POSTN by hPDL Cells.

PubMed

Manokawinchoke, J; Limjeerajarus, N; Limjeerajarus, C; Sastravaha, P; Everts, V; Pavasant, P

2015-07-01

The aim of this study was to investigate the response of human periodontal ligament (hPDL) fibroblasts to an intermittent compressive force and its effect on the expression of SOST, POSTN, and TGFB1. A computerized cell compressive force loading apparatus was introduced, and hPDL cells were subjected to intermittent compressive force. The changes in messenger RNA (mRNA) and protein expression were monitored by real-time polymerase chain reaction and Western blot analysis, respectively. An increased expression of SOST, POSTN, and TGFB1 was observed in a time-dependent fashion. Addition of cycloheximide, a transforming growth factor (TGF)-β inhibitor (SB431542), or a neutralizing antibody against TGF-β1 attenuated the force-induced expression of SOST and POSTN as well as sclerostin and periostin, indicating a role of TGF-β1 in the pressure-induced expression of these proteins. Enzyme-linked immunosorbent assay analysis revealed an increased level of TGF-β1 in the cell extracts but not in the medium, suggesting that intermittent compressive force promoted the accumulation of TGF-β1 in the cells or their surrounding matrix. In conclusion, an intermittent compressive force regulates SOST/POSTN expression by hPDL cells via the TGF-β1 signaling pathway. Since these proteins play important roles in the homeostasis of the periodontal tissue, our results indicate the importance of masticatory forces in this process. © International & American Associations for Dental Research 2015.

Nine unanswered questions about cytokinesis

PubMed Central

2017-01-01

Experiments on model systems have revealed that cytokinesis in cells with contractile rings (amoebas, fungi, and animals) depends on shared molecular mechanisms in spite of some differences that emerged during a billion years of divergent evolution. Understanding these fundamental mechanisms depends on identifying the participating proteins and characterizing the mechanisms that position the furrow, assemble the contractile ring, anchor the ring to the plasma membrane, trigger ring constriction, produce force to form a furrow, disassemble the ring, expand the plasma membrane in the furrow, and separate the daughter cell membranes. This review reveals that fascinating questions remain about each step. PMID:28807993

Nine unanswered questions about cytokinesis.

PubMed

Pollard, Thomas D

2017-10-02

Experiments on model systems have revealed that cytokinesis in cells with contractile rings (amoebas, fungi, and animals) depends on shared molecular mechanisms in spite of some differences that emerged during a billion years of divergent evolution. Understanding these fundamental mechanisms depends on identifying the participating proteins and characterizing the mechanisms that position the furrow, assemble the contractile ring, anchor the ring to the plasma membrane, trigger ring constriction, produce force to form a furrow, disassemble the ring, expand the plasma membrane in the furrow, and separate the daughter cell membranes. This review reveals that fascinating questions remain about each step. © 2017 Pollard.

Organization of supercoil domains and their reorganization by transcription

PubMed Central

Deng, Shuang; Stein, Richard A.; Higgins, N. Patrick

2006-01-01

Summary During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require specific types of DNA movement. Separation forces move replicated chromosomes into separate sister cell compartments during cell division, and the contemporaneous acts of DNA replication, RNA transcription and cotranscriptional translation of membrane proteins cause specific regions of DNA to twist, writhe and expand or contract. Recent experiments indicate that a dynamic and stochastic mechanism creates supercoil DNA domains soon after DNA replication. Domain structure is subsequently reorganized by RNA transcription. Examples of transcription-dependent chromosome remodelling are also emerging from eukaryotic cell systems. PMID:16135220

α-Helix Unwinding as Force Buffer in Spectrins.

PubMed

Takahashi, Hirohide; Rico, Felix; Chipot, Christophe; Scheuring, Simon

2018-03-27

Spectrins are cytoskeletal proteins located at the inner face of the plasma membrane, making connections between membrane anchors and the actin cortex, and between actin filaments. Spectrins share a common structure forming a bundle of 3 α-helices and play a major role during cell deformation. Here, we used high-speed force spectroscopy and steered molecular dynamics simulations to understand the mechanical stability of spectrin, revealing a molecular force buffering function. We find that spectrin acts as a soft spring at short extensions (70-100 Å). Under continuous external stretching, its α-helices unwind, leading to a viscous mechanical response over larger extensions (100-300 Å), represented by a constant-force plateau in force/extension curves. This viscous force buffering emerges from a quasi-equilibrium competition between disruption and re-formation of α-helical hydrogen bonds. Our results suggest that, in contrast to β-sheet proteins, which unfold in a catastrophic event, α-helical spectrins dominantly unwind, providing a viscous force buffer over extensions about 5 times their folded length.

Effects of different force fields on the structural character of α synuclein β-hairpin peptide (35-56) in aqueous environment.

PubMed

Kundu, Sangeeta

2018-02-01

The hallmark of Parkinson's disease (PD) is the intracellular protein aggregation forming Lewy Bodies (LB) and Lewy neuritis which comprise mostly of a protein, alpha synuclein (α-syn). Molecular dynamics (MD) simulation methods can augment experimental techniques to understand misfolding and aggregation pathways with atomistic resolution. The quality of MD simulations for proteins and peptides depends greatly on the accuracy of empirical force fields. The aim of this work is to investigate the effects of different force fields on the structural character of β hairpin fragment of α-syn (residues 35-56) peptide in aqueous solution. Six independent MD simulations are done in explicit solvent using, AMBER03, AMBER99SB, GROMOS96 43A1, GROMOS96 53A6, OPLS-AA, and CHARMM27 force fields with CMAP corrections. The performance of each force field is assessed from several structural parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), formation of β-turn, the stability of folded β-hairpin structure, and the favourable conformations obtained for different force fields. In this study, CMAP correction of CHARMM27 force field is found to overestimate the helical conformation, while GROMOS96 53A6 is found to most successfully capture the conformational dynamics of α-syn β-hairpin fragment as elicited from NMR.

Electrochemically controlled drug-mimicking protein release from iron-alginate thin-films associated with an electrode.

PubMed

Jin, Zhiyuan; Güven, Güray; Bocharova, Vera; Halámek, Jan; Tokarev, Ihor; Minko, Sergiy; Melman, Artem; Mandler, Daniel; Katz, Evgeny

2012-01-01

Novel biocompatible hybrid-material composed of iron-ion-cross-linked alginate with embedded protein molecules has been designed for the signal-triggered drug release. Electrochemically controlled oxidation of Fe(2+) ions in the presence of soluble natural alginate polymer and drug-mimicking protein (bovine serum albumin, BSA) results in the formation of an alginate-based thin-film cross-linked by Fe(3+) ions at the electrode interface with the entrapped protein. The electrochemically generated composite thin-film was characterized by electrochemistry and atomic force microscopy (AFM). Preliminary experiments demonstrated that the electrochemically controlled deposition of the protein-containing thin-film can be performed at microscale using scanning electrochemical microscopy (SECM) as the deposition tool producing polymer-patterned spots potentially containing various entrapped drugs. Application of reductive potentials on the modified electrode produced Fe(2+) cations which do not keep complexation with alginate, thus resulting in the electrochemically triggered thin-film dissolution and the protein release. Different experimental parameters, such as the film-deposition time, concentrations of compounds and applied potentials, were varied in order to demonstrate that the electrodepositon and electrodissolution of the alginate composite film can be tuned to the optimum performance. A statistical modeling technique was applied to find optimal conditions for the formation of the composite thin-film for the maximal encapsulation and release of the drug-mimicking protein at the lowest possible potential. © 2011 American Chemical Society

Exploring the parameter space of the coarse-grained UNRES force field by random search: selecting a transferable medium-resolution force field.

PubMed

He, Yi; Xiao, Yi; Liwo, Adam; Scheraga, Harold A

2009-10-01

We explored the energy-parameter space of our coarse-grained UNRES force field for large-scale ab initio simulations of protein folding, to obtain good initial approximations for hierarchical optimization of the force field with new virtual-bond-angle bending and side-chain-rotamer potentials which we recently introduced to replace the statistical potentials. 100 sets of energy-term weights were generated randomly, and good sets were selected by carrying out replica-exchange molecular dynamics simulations of two peptides with a minimal alpha-helical and a minimal beta-hairpin fold, respectively: the tryptophan cage (PDB code: 1L2Y) and tryptophan zipper (PDB code: 1LE1). Eight sets of parameters produced native-like structures of these two peptides. These eight sets were tested on two larger proteins: the engrailed homeodomain (PDB code: 1ENH) and FBP WW domain (PDB code: 1E0L); two sets were found to produce native-like conformations of these proteins. These two sets were tested further on a larger set of nine proteins with alpha or alpha + beta structure and found to locate native-like structures of most of them. These results demonstrate that, in addition to finding reasonable initial starting points for optimization, an extensive search of parameter space is a powerful method to produce a transferable force field. Copyright 2009 Wiley Periodicals, Inc.

Robust mechanobiological behavior emerges in heterogeneous myosin systems.

PubMed

Egan, Paul F; Moore, Jeffrey R; Ehrlicher, Allen J; Weitz, David A; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

2017-09-26

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

Robust mechanobiological behavior emerges in heterogeneous myosin systems

NASA Astrophysics Data System (ADS)

Egan, Paul F.; Moore, Jeffrey R.; Ehrlicher, Allen J.; Weitz, David A.; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

2017-09-01

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

Microgravity

NASA Image and Video Library

2001-06-06

Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

Influence of structure properties on protein-protein interactions-QSAR modeling of changes in diffusion coefficients.

PubMed

Bauer, Katharina Christin; Hämmerling, Frank; Kittelmann, Jörg; Dürr, Cathrin; Görlich, Fabian; Hubbuch, Jürgen

2017-04-01

Information about protein-protein interactions provides valuable knowledge about the phase behavior of protein solutions during the biopharmaceutical production process. Up to date it is possible to capture their overall impact by an experimentally determined potential of mean force. For the description of this potential, the second virial coefficient B22, the diffusion interaction parameter kD, the storage modulus G', or the diffusion coefficient D is applied. In silico methods do not only have the potential to predict these parameters, but also to provide deeper understanding of the molecular origin of the protein-protein interactions by correlating the data to the protein's three-dimensional structure. This methodology furthermore allows a lower sample consumption and less experimental effort. Of all in silico methods, QSAR modeling, which correlates the properties of the molecule's structure with the experimental behavior, seems to be particularly suitable for this purpose. To verify this, the study reported here dealt with the determination of a QSAR model for the diffusion coefficient of proteins. This model consisted of diffusion coefficients for six different model proteins at various pH values and NaCl concentrations. The generated QSAR model showed a good correlation between experimental and predicted data with a coefficient of determination R2 = 0.9 and a good predictability for an external test set with R2 = 0.91. The information about the properties affecting protein-protein interactions present in solution was in agreement with experiment and theory. Furthermore, the model was able to give a more detailed picture of the protein properties influencing the diffusion coefficient and the acting protein-protein interactions. Biotechnol. Bioeng. 2017;114: 821-831. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Improved model of hydrated calcium ion for molecular dynamics simulations using classical biomolecular force fields.

PubMed

Yoo, Jejoong; Wilson, James; Aksimentiev, Aleksei

2016-10-01

Calcium ions (Ca(2+) ) play key roles in various fundamental biological processes such as cell signaling and brain function. Molecular dynamics (MD) simulations have been used to study such interactions, however, the accuracy of the Ca(2+) models provided by the standard MD force fields has not been rigorously tested. Here, we assess the performance of the Ca(2+) models from the most popular classical force fields AMBER and CHARMM by computing the osmotic pressure of model compounds and the free energy of DNA-DNA interactions. In the simulations performed using the two standard models, Ca(2+) ions are seen to form artificial clusters with chloride, acetate, and phosphate species; the osmotic pressure of CaAc2 and CaCl2 solutions is a small fraction of the experimental values for both force fields. Using the standard parameterization of Ca(2+) ions in the simulations of Ca(2+) -mediated DNA-DNA interactions leads to qualitatively wrong outcomes: both AMBER and CHARMM simulations suggest strong inter-DNA attraction whereas, in experiment, DNA molecules repel one another. The artificial attraction of Ca(2+) to DNA phosphate is strong enough to affect the direction of the electric field-driven translocation of DNA through a solid-state nanopore. To address these shortcomings of the standard Ca(2+) model, we introduce a custom model of a hydrated Ca(2+) ion and show that using our model brings the results of the above MD simulations in quantitative agreement with experiment. Our improved model of Ca(2+) can be readily applied to MD simulations of various biomolecular systems, including nucleic acids, proteins and lipid bilayer membranes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 752-763, 2016. © 2016 Wiley Periodicals, Inc.

Role of TRPC1 channel in skeletal muscle function

PubMed Central

Zanou, Nadège; Shapovalov, Georges; Louis, Magali; Tajeddine, Nicolas; Gallo, Chiara; Van Schoor, Monique; Anguish, Isabelle; Cao, My Linh; Schakman, Olivier; Dietrich, Alexander; Lebacq, Jean; Ruegg, Urs; Roulet, Emmanuelle; Birnbaumer, Lutz

2010-01-01

Skeletal muscle contraction is reputed not to depend on extracellular Ca2+. Indeed, stricto sensu, excitation-contraction coupling does not necessitate entry of Ca2+. However, we previously observed that, during sustained activity (repeated contractions), entry of Ca2+ is needed to maintain force production. In the present study, we evaluated the possible involvement of the canonical transient receptor potential (TRPC)1 ion channel in this entry of Ca2+ and investigated its possible role in muscle function. Patch-clamp experiments reveal the presence of a small-conductance channel (13 pS) that is completely lost in adult fibers from TRPC1−/− mice. The influx of Ca2+ through TRPC1 channels represents a minor part of the entry of Ca2+ into muscle fibers at rest, and the activity of the channel is not store dependent. The lack of TRPC1 does not affect intracellular Ca2+ concentration ([Ca2+]i) transients reached during a single isometric contraction. However, the involvement of TRPC1-related Ca2+ entry is clearly emphasized in muscle fatigue. Indeed, muscles from TRPC1−/− mice stimulated repeatedly progressively display lower [Ca2+]i transients than those observed in TRPC1+/+ fibers, and they also present an accentuated progressive loss of force. Interestingly, muscles from TRPC1−/− mice display a smaller fiber cross-sectional area, generate less force per cross-sectional area, and contain less myofibrillar proteins than their controls. They do not present other signs of myopathy. In agreement with in vitro experiments, TRPC1−/− mice present an important decrease of endurance of physical activity. We conclude that TRPC1 ion channels modulate the entry of Ca2+ during repeated contractions and help muscles to maintain their force during sustained repeated contractions. PMID:19846750

Theoretical analysis for the design of the French watt balance experiment force comparator

NASA Astrophysics Data System (ADS)

Pinot, Patrick; Genevès, Gerard; Haddad, Darine; David, Jean; Juncar, Patrick; Lecollinet, Michel; Macé, Stéphane; Villar, François

2007-09-01

This paper presents a preliminary analysis for designing a force comparator to be used in the French watt balance experiment. The first stage of this experiment consists in a static equilibrium, by means of a mechanical beam balance, between a gravitational force (a weight of an artefact having a known mass submitted to the acceleration due to the gravity) and a vertical electromagnetic force acting on a coil driven by a current subject to the magnetic induction field provided by a permanent magnet. The principle of the force comparison in the French experiment is explained. The general design configuration of the force balance using flexure strips as pivots is discussed and theoretical calculation results based on realistic assumptions of the static and dynamic behaviors of the balance are presented.

Theoretical analysis for the design of the French watt balance experiment force comparator.

PubMed

Pinot, Patrick; Genevès, Gerard; Haddad, Darine; David, Jean; Juncar, Patrick; Lecollinet, Michel; Macé, Stéphane; Villar, François

2007-09-01

This paper presents a preliminary analysis for designing a force comparator to be used in the French watt balance experiment. The first stage of this experiment consists in a static equilibrium, by means of a mechanical beam balance, between a gravitational force (a weight of an artefact having a known mass submitted to the acceleration due to the gravity) and a vertical electromagnetic force acting on a coil driven by a current subject to the magnetic induction field provided by a permanent magnet. The principle of the force comparison in the French experiment is explained. The general design configuration of the force balance using flexure strips as pivots is discussed and theoretical calculation results based on realistic assumptions of the static and dynamic behaviors of the balance are presented.

Consistent View of Protein Fluctuations from All-Atom Molecular Dynamics and Coarse-Grained Dynamics with Knowledge-Based Force-Field.

PubMed

Jamroz, Michal; Orozco, Modesto; Kolinski, Andrzej; Kmiecik, Sebastian

2013-01-08

It is widely recognized that atomistic Molecular Dynamics (MD), a classical simulation method, captures the essential physics of protein dynamics. That idea is supported by a theoretical study showing that various MD force-fields provide a consensus picture of protein fluctuations in aqueous solution [Rueda, M. et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 796-801]. However, atomistic MD cannot be applied to most biologically relevant processes due to its limitation to relatively short time scales. Much longer time scales can be accessed by properly designed coarse-grained models. We demonstrate that the aforementioned consensus view of protein dynamics from short (nanosecond) time scale MD simulations is fairly consistent with the dynamics of the coarse-grained protein model - the CABS model. The CABS model employs stochastic dynamics (a Monte Carlo method) and a knowledge-based force-field, which is not biased toward the native structure of a simulated protein. Since CABS-based dynamics allows for the simulation of entire folding (or multiple folding events) in a single run, integration of the CABS approach with all-atom MD promises a convenient (and computationally feasible) means for the long-time multiscale molecular modeling of protein systems with atomistic resolution.

A random walk in physical biology

NASA Astrophysics Data System (ADS)

Peterson, Eric Lee

Biology as a scientific discipline is becoming evermore quantitative as tools become available to probe living systems on every scale from the macro to the micro and now even to the nanoscale. In quantitative biology the challenge is to understand the living world in an in vivo context, where it is often difficult for simple theoretical models to connect with the full richness and complexity of the observed data. Computational models and simulations offer a way to bridge the gap between simple theoretical models and real biological systems; towards that aspiration are presented in this thesis three case studies in applying computational models that may give insight into native biological structures.The first is concerned with soluble proteins; proteins, like DNA, are linear polymers written in a twenty-letter "language" of amino acids. Despite the astronomical number of possible proteins sequences, a great amount of similarity is observed among the folded structures of globular proteins. One useful way of discovering similar sequences is to align their sequences, as done e.g. by the popular BLAST program. By clustering together amino acids and reducing the alphabet that proteins are written in to fewer than twenty letters, we find that pairwise sequence alignments are actually more sensitive to proteins with similar structures.The second case study is concerned with the measurement of forces applied to a membrane. We demonstrate a general method for extracting the forces applied to a fluid lipid bilayer of arbitrary shape and show that the subpiconewton forces applied by optical tweezers to vesicles can be accurately measured in this way.In the third and final case study we examine the forces between proteins in a lipid bilayer membrane. Due to the bending of the membrane surrounding them, such proteins feel mutually attractive forces which can help them to self-organize and act in concert. These finding are relevant at the areal densities estimated for membrane proteins such as the MscL mechanosensitive channel. The findings of the analytical studies were confirmed by a Monte Carlo Markov Chain simulation using the fully two-dimensional potentials between two model proteins in a membrane.Living systems present us with beautiful and intricate structures, from the helices and sheets of a folded protein to the dynamic morphology of cellular organelles and the self-organization of proteins in a biomembrane and a synergy of theoretical and it in silico approaches should enable us to build and refine models of in vivo biological data.

Force measurements in stiff, 3D, opaque granular materials

NASA Astrophysics Data System (ADS)

Hurley, Ryan C.; Hall, Stephen A.; Andrade, José E.; Wright, Jonathan

2017-06-01

We present results from two experiments that provide the first quantification of inter-particle force networks in stiff, 3D, opaque granular materials. Force vectors between all grains were determined using a mathematical optimization technique that seeks to satisfy grain equilibrium and strain measurements. Quantities needed in the optimization - the spatial location of the inter-particle contact network and tensor grain strains - were found using 3D X-ray diffraction and X-ray computed tomography. The statistics of the force networks are consistent with those found in past simulations and 2D experiments. In particular, we observe an exponential decay of normal forces above the mean and a partition of forces into strong and weak networks. In the first experiment, involving 77 single-crystal quartz grains, we also report on the temporal correlation of the force network across two sequential load cycles. In the second experiment, involving 1099 single-crystal ruby grains, we characterize force network statistics at low levels of compression.

Protein-Backbone Thermodynamics across the Membrane Interface.

PubMed

Bereau, Tristan; Kremer, Kurt

2016-07-07

The thermodynamics of insertion of a protein in a membrane depends on the fine interplay between backbone and side-chain contributions interacting with the lipid environment. Using computer simulations, we probe how different descriptions of the backbone glycyl unit affect the thermodynamics of insertion of individual residues, dipeptides, and entire transmembrane helices. Due to the lack of reference data, we first introduce an efficient methodology to estimate atomistic potential of mean force (PMF) curves from a series of representative and uncorrelated coarse-grained (CG) snapshots. We find strong discrepancies between two CG models, Martini and PLUM, against reference atomistic PMFs and experiments. Atomistic simulations suggest a weak free energy of insertion between water and a POPC membrane for the glycyl unit, in overall agreement with experimental results despite severe assumptions in our calculations. We show that refining the backbone contribution in PLUM significantly improves the PMF of insertion of the WALP16 transmembrane peptide. An improper balance between the glycyl backbone and the attached side chain will lead to energetic artifacts, rationalizing Martini's overstabilization of WALP's adsorbed interfacial state. It illustrates difficulties associated with free-energy-based parametrizations of single-residue models, as the relevant free energy of partitioning used for force-field parametrization does not arise from the entire residue but rather the solvent-accessible chemical groups.

Impact of the alkyl chain length on binding of imidazolium-based ionic liquids to bovine serum albumin

NASA Astrophysics Data System (ADS)

Zhang, Mengyue; Wang, Ying; Zhang, Hongmei; Cao, Jian; Fei, Zhenghao; Wang, Yanqing

2018-05-01

The effects of six imidazolium-based ionic liquids (ILs) with different alkyl chain length ([CnMim]Cl, n = 2, 4, 6, 8, 10, 12) on the structure and functions of bovine serum albumin (BSA) were studied by multi-spectral methods and molecular docking. ILs with the longer alkyl chain length have the stronger binding interaction with BSA and the greater conformational damage to protein. The effects of ILs on the functional properties of BSA were further studied by the determination of non-enzyme esterase activity, β-fibrosis and other properties of BSA. The thermal stability of BSA was reduced, the rate of the formation of beta sheet structures of BSA was lowered, and the esterase-like activity of BSA were decreased with the increase of ILs concentration. Simultaneous molecular modeling technique revealed the favorable binding sites of ILs on protein. The hydrophobic force and polar interactions were the mainly binding forces of them. The calculated results are in a good agreement with the spectroscopic experiments. These studies on the impact of the alkyl chain length on binding of imidazolium-based ionic liquids to BSA are of great significance for understanding and developing the application of ionic liquid in life and physiological system.

Ultrastable cellulosome-adhesion complex tightens under load

PubMed Central

Schoeler, Constantin; Malinowska, Klara H.; Bernardi, Rafael C.; Milles, Lukas F.; Jobst, Markus A.; Durner, Ellis; Ott, Wolfgang; Fried, Daniel B.; Bayer, Edward A.; Schulten, Klaus; Gaub, Hermann E.; Nash, Michael A.

2014-01-01

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand–receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand–receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600–750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass. PMID:25482395

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

PubMed

Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

2014-12-18

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fertilization of sea urchin eggs and sperm motility are negatively impacted under low hypergravitational forces significant to space flight

NASA Technical Reports Server (NTRS)

Tash, J. S.; Kim, S.; Schuber, M.; Seibt, D.; Kinsey, W. H.

2001-01-01

Sperm and other flagellates swim faster in microgravity (microG) than in 1 G, raising the question of whether fertilization is altered under conditions of space travel. Such alterations have implications for reproduction of plant and animal food and for long-term space habitation by man. We previously demonstrated that microG accelerates protein phosphorylation during initiation of sperm motility but delays the sperm response to the egg chemotactic factor, speract. Thus sperm are sensitive to changes in gravitational force. New experiments using the NiZeMi centrifugal microscope examined whether low hypergravity (hyperG) causes effects opposite to microG on sperm motility, signal transduction, and fertilization. Sperm % motility and straight-line velocity were significantly inhibited by as little as 1.3 G. The phosphorylation states of FP130, an axonemal phosphoprotein, and FP160, a cAMP-dependent salt-extractable flagellar protein, both coupled to motility activation, showed a more rapid decline in hyperG. Most critically, hyperG caused an approximately 50% reduction in both the rate of sperm-egg binding and fertilization. The similar extent of inhibition of both fertilization parameters in hyperG suggests that the primary effect is on sperm rather than eggs. These results not only support our earlier microG data demonstrating that sperm are sensitive to small changes in gravitational forces but more importantly now show that this sensitivity affects the ability of sperm to fertilize eggs. Thus, more detailed studies on the impact of space flight on development should include studies of sperm function and fertilization.

Directly measuring single-molecule heterogeneity using force spectroscopy

PubMed Central

Hinczewski, Michael; Thirumalai, D.

2016-01-01

One of the most intriguing results of single-molecule experiments on proteins and nucleic acids is the discovery of functional heterogeneity: the observation that complex cellular machines exhibit multiple, biologically active conformations. The structural differences between these conformations may be subtle, but each distinct state can be remarkably long-lived, with interconversions between states occurring only at macroscopic timescales, fractions of a second or longer. Although we now have proof of functional heterogeneity in a handful of systems—enzymes, motors, adhesion complexes—identifying and measuring it remains a formidable challenge. Here, we show that evidence of this phenomenon is more widespread than previously known, encoded in data collected from some of the most well-established single-molecule techniques: atomic force microscopy or optical tweezer pulling experiments. We present a theoretical procedure for analyzing distributions of rupture/unfolding forces recorded at different pulling speeds. This results in a single parameter, quantifying the degree of heterogeneity, and also leads to bounds on the equilibration and conformational interconversion timescales. Surveying 10 published datasets, we find heterogeneity in 5 of them, all with interconversion rates slower than 10 s−1. Moreover, we identify two systems where additional data at realizable pulling velocities is likely to find a theoretically predicted, but so far unobserved crossover regime between heterogeneous and nonheterogeneous behavior. The significance of this regime is that it will allow far more precise estimates of the slow conformational switching times, one of the least understood aspects of functional heterogeneity. PMID:27317744

Biophysical properties of intrinsically disordered p130Cas substrate domain--implication in mechanosensing.

PubMed

Hotta, Kinya; Ranganathan, Soumya; Liu, Ruchuan; Wu, Fei; Machiyama, Hiroaki; Gao, Rong; Hirata, Hiroaki; Soni, Neelesh; Ohe, Takashi; Hogue, Christopher W V; Madhusudhan, M S; Sawada, Yasuhiro

2014-04-01

Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2-9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.

EDITORIAL: Nanotechnology at the interface of cell biology, materials science and medicine Nanotechnology at the interface of cell biology, materials science and medicine

NASA Astrophysics Data System (ADS)

Engel, Andreas; Miles, Mervyn

2008-09-01

The atomic force microscope (AFM) and related scanning probe microscopes have become resourceful tools to study cells, supramolecular assemblies and single biomolecules, because they allow investigations of such structures in native environments. Quantitative information has been gathered about the surface structure of membrane proteins to lateral and vertical resolutions of 0.5 nm and 0.1 nm, respectively, about the forces that keep protein-protein and protein-nucleic acid assemblies together as well as single proteins in their native conformation, and about the nanomechanical properties of cells in health and disease. Such progress has been achieved mainly because of constant development of AFM instrumentation and sample preparation methods. This special issue of Nanotechnology presents papers from leading laboratories in the field of nanobiology, covering a wide range of topics in the form of original and novel scientific contributions. It addresses achievements in instrumentation, sample preparation, automation and in biological applications. These papers document the creativity and persistence of researchers pursuing the goal to unravel the structure and dynamics of cells, supramolecuar structures and single biomolecules at work. Improved cantilever sensors, novel optical probes, and quantitative data on supports for electrochemical experiments open new avenues for characterizing biological nanomachines down to the single molecule. Comparative measurements of healthy and metastatic cells promise new methods for early detection of tumors, and possible assessments of drug efficacy. High-speed AFMs document possibilities to monitor crystal growth and to observe large structures at video rate. A wealth of information on amyloid-type fibers as well as on membrane proteins has been gathered by single molecule force spectroscopy—a technology now being automated for large-scale data collection. With the progress of basic research and a strong industry supporting instrumentation development by improving robustness and reliability and making new instruments available to the community, nanobiology has the potential to develop into a field with great impact on our understanding of the complexity of life, and to provide a major contribution to human health. This special issue of Nanotechnology on nanobiology would not have been possible without the highly professional support from Nina Couzin, Amy Harvey and the Nanotechnology team at IOP Publishing. We are thankful for their most constructive and effective help in pushing the project forward. We are also thankful to all the authors who have contributed with excellent original articles, as well as to the referees who have helped to make this special issue such an insightful document of a rapidly moving field.

Sequential protein unfolding through a carbon nanotube pore

NASA Astrophysics Data System (ADS)

Xu, Zhonghe; Zhang, Shuang; Weber, Jeffrey K.; Luan, Binquan; Zhou, Ruhong; Li, Jingyuan

2016-06-01

An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable ``unfoldon'' motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability.An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable ``unfoldon'' motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00410e

Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1α

PubMed Central

2014-01-01

Background Endoplasmic reticulum stress, caused by the presence of misfolded proteins, activates the stress sensor inositol-requiring enzyme 1α (IRE1α). The resulting increase in IRE1α RNase activity causes sequence-specific cleavage of X-box binding protein 1 (XBP1) mRNA, resulting in upregulation of the unfolded protein response and cellular adaptation to stress. The precise mechanism of human IRE1α activation is currently unclear. The role of IRE1α kinase activity is disputed, as results from the generation of various kinase-inactivating mutations in either yeast or human cells are discordant. Kinase activity can also be made redundant by small molecules which bind the ATP binding site. We set out to uncover a role for IRE1α kinase activity using wild-type cytosolic protein constructs. Results We show that concentration-dependent oligomerisation is sufficient to cause IRE1α cytosolic domain RNase activity in vitro. We demonstrate a role for the kinase activity by showing that autophosphorylation enhances RNase activity. Inclusion of the IRE1α linker domain in protein constructs allows hyperphosphorylation and further enhancement of RNase activity, highlighting the importance of kinase activity. We show that IRE1α phosphorylation status correlates with an increased propensity to form oligomeric complexes and that forced dimerisation causes great enhancement in RNase activity. In addition we demonstrate that even when IRE1α is forced to dimerise, by a GST-tag, phospho-enhancement of activity is still observed. Conclusions Taken together these experiments support the hypothesis that phosphorylation is important in modulating IRE1α RNase activity which is achieved by increasing the propensity of IRE1α to dimerise. This work supports the development of IRE1α kinase inhibitors for use in the treatment of secretory cancers. PMID:24524643

Force feedback effects on single molecule hopping and pulling experiments

NASA Astrophysics Data System (ADS)

Rico-Pasto, M.; Pastor, I.; Ritort, F.

2018-03-01

Single-molecule experiments with optical tweezers have become an important tool to study the properties and mechanisms of biological systems, such as cells and nucleic acids. In particular, force unzipping experiments have been used to extract the thermodynamics and kinetics of folding and unfolding reactions. In hopping experiments, a molecule executes transitions between the unfolded and folded states at a preset value of the force [constant force mode (CFM) under force feedback] or trap position [passive mode (PM) without feedback] and the force-dependent kinetic rates extracted from the lifetime of each state (CFM) and the rupture force distributions (PM) using the Bell-Evans model. However, hopping experiments in the CFM are known to overestimate molecular distances and folding free energies for fast transitions compared to the response time of the feedback. In contrast, kinetic rate measurements from pulling experiments have been mostly done in the PM while the CFM is seldom implemented in pulling protocols. Here, we carry out hopping and pulling experiments in a short DNA hairpin in the PM and CFM at three different temperatures (6 °C, 25 °C, and 45 °C) exhibiting largely varying kinetic rates. As expected, we find that equilibrium hopping experiments in the CFM and PM perform well at 6 °C (where kinetics are slow), whereas the CFM overestimates molecular parameters at 45 °C (where kinetics are fast). In contrast, nonequilibrium pulling experiments perform well in both modes at all temperatures. This demonstrates that the same kind of feedback algorithm in the CFM leads to more reliable determination of the folding reaction parameters in irreversible pulling experiments.

Force feedback effects on single molecule hopping and pulling experiments.

PubMed

Rico-Pasto, M; Pastor, I; Ritort, F

2018-03-28

Single-molecule experiments with optical tweezers have become an important tool to study the properties and mechanisms of biological systems, such as cells and nucleic acids. In particular, force unzipping experiments have been used to extract the thermodynamics and kinetics of folding and unfolding reactions. In hopping experiments, a molecule executes transitions between the unfolded and folded states at a preset value of the force [constant force mode (CFM) under force feedback] or trap position [passive mode (PM) without feedback] and the force-dependent kinetic rates extracted from the lifetime of each state (CFM) and the rupture force distributions (PM) using the Bell-Evans model. However, hopping experiments in the CFM are known to overestimate molecular distances and folding free energies for fast transitions compared to the response time of the feedback. In contrast, kinetic rate measurements from pulling experiments have been mostly done in the PM while the CFM is seldom implemented in pulling protocols. Here, we carry out hopping and pulling experiments in a short DNA hairpin in the PM and CFM at three different temperatures (6 °C, 25 °C, and 45 °C) exhibiting largely varying kinetic rates. As expected, we find that equilibrium hopping experiments in the CFM and PM perform well at 6 °C (where kinetics are slow), whereas the CFM overestimates molecular parameters at 45 °C (where kinetics are fast). In contrast, nonequilibrium pulling experiments perform well in both modes at all temperatures. This demonstrates that the same kind of feedback algorithm in the CFM leads to more reliable determination of the folding reaction parameters in irreversible pulling experiments.

Initial Flight Tests of the NASA F-15B Propulsion Flight Test Fixture

NASA Technical Reports Server (NTRS)

Palumbo, Nathan; Moes, Timothy R.; Vachon, M. Jake

2002-01-01

Flights of the F-15B/Propulsion Flight Test Fixture (PFTF) with a Cone Drag Experiment (CDE) attached have been accomplished at NASA Dryden Flight Research Center. Mounted underneath the fuselage of an F-15B airplane, the PFTF provides volume for experiment systems and attachment points for propulsion experiments. A unique feature of the PFTF is the incorporation of a six-degree-of-freedom force balance. The force balance mounts between the PFTF and experiment and measures three forces and moments. The CDE has been attached to the force balance for envelope expansion flights. This experiment spatially and inertially simulates a large propulsion test article. This report briefly describes the F-15B airplane, the PFTF, and the force balance. A detailed description of the CDE is provided. Force-balance ground testing and stiffness modifications are described. Flight profiles and selected flight data from the envelope expansion flights are provided and discussed, including force-balance data, the internal PFTF thermal and vibration environment, a handling qualities assessment, and performance capabilities of the F-15B airplane with the PFTF installed.

Interplay of protein and DNA structure revealed in simulations of the lac operon.

PubMed

Czapla, Luke; Grosner, Michael A; Swigon, David; Olson, Wilma K

2013-01-01

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

PubMed Central

Suhr, Matthias; Raff, Johannes; Pollmann, Katrin

2016-01-01

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding. The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces. PMID:26863150

Differences in Contractile Function of Myofibrils within Human Embryonic Stem Cell-Derived Cardiomyocytes vs. Adult Ventricular Myofibrils Are Related to Distinct Sarcomeric Protein Isoforms

PubMed Central

Iorga, Bogdan; Schwanke, Kristin; Weber, Natalie; Wendland, Meike; Greten, Stephan; Piep, Birgit; dos Remedios, Cristobal G.; Martin, Ulrich; Zweigerdt, Robert; Kraft, Theresia; Brenner, Bernhard

2018-01-01

Characterizing the contractile function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is key for advancing their utility for cellular disease models, promoting cell based heart repair, or developing novel pharmacological interventions targeting cardiac diseases. The aim of the present study was to understand whether steady-state and kinetic force parameters of β-myosin heavy chain (βMyHC) isoform-expressing myofibrils within human embryonic stem cell-derived cardiomyocytes (hESC-CMs) differentiated in vitro resemble those of human ventricular myofibrils (hvMFs) isolated from adult donor hearts. Contractile parameters were determined using the same micromechanical method and experimental conditions for both types of myofibrils. We identified isoforms and phosphorylation of main sarcomeric proteins involved in the modulation of force generation of both, chemically demembranated hESC-CMs (d-hESC-CMs) and hvMFs. Our results indicate that at saturating Ca2+ concentration, both human-derived contractile systems developed forces with similar rate constants (0.66 and 0.68 s−1), reaching maximum isometric force that was significantly smaller for d-hESC-CMs (42 kPa) than for hvMFs (94 kPa). At submaximal Ca2+-activation, where intact cardiomyocytes normally operate, contractile parameters of d-hESC-CMs and hvMFs exhibited differences. Ca2+ sensitivity of force was higher for d-hESC-CMs (pCa50 = 6.04) than for hvMFs (pCa50 = 5.80). At half-maximum activation, the rate constant for force redevelopment was significantly faster for d-hESC-CMs (0.51 s−1) than for hvMFs (0.28 s−1). During myofibril relaxation, kinetics of the slow force decay phase were significantly faster for d-hESC-CMs (0.26 s−1) than for hvMFs (0.21 s−1), while kinetics of the fast force decay were similar and ~20x faster. Protein analysis revealed that hESC-CMs had essentially no cardiac troponin-I, and partially non-ventricular isoforms of some other sarcomeric proteins, explaining the functional discrepancies. The sarcomeric protein isoform pattern of hESC-CMs had features of human cardiomyocytes at an early developmental stage. The study indicates that morphological and ultrastructural maturation of βMyHC isoform-expressing hESC-CMs is not necessarily accompanied by ventricular-like expression of all sarcomeric proteins. Our data suggest that hPSC-CMs could provide useful tools for investigating inherited cardiac diseases affecting contractile function during early developmental stages. PMID:29403388

Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

PubMed

Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

2014-08-01

The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel approach for application of nylon membranes in the biosensing domain

NASA Astrophysics Data System (ADS)

Farahmand, Elham; Ibrahim, Fatimah; Hosseini, Samira; Rothan, Hussin A.; Yusof, Rohana; Koole, Leo H.; Djordjevic, Ivan

2015-10-01

In this paper we report the polymer-coated microporous nylon membranes and their application as platforms for protein immobilization and subsequent detection of the dengue virus (DV) in blood serum. Protein recognition experiments were performed with enzyme-linked immunosorbent assay (ELISA). The polymers used for coatings were synthesized by free-radical polymerization reaction between methyl methacrylate (MMA) and methacrylic acid (MAA) in different concentrations. The MAA monomer has carefully been chosen to generate polymers with pendant carboxyl (-COOH) groups, which also exist on polymer surfaces. A high degree of control over surface-exposed -COOH groups has been achieved through variation of monomers concentration in polymerization reaction. The general aspect of this work relies on the dengue antibody (Ab) immobilization on surface -COOH groups via physical attachment or covalent immobilization. Prior to Ab immobilization and ELISA experiment, polymer-coated nylon samples were analyzed in detail for their physical properties by atomic force microscopy (AFM), scanning electron microscopy (SEM), and water-in-air contact angle (WCA) measurements. Membranes were further analyzed by Fourier transform infrared spectroscopy (FTIR) in order to establish the relationship between wettability, porosity, and surface roughness with chemical composition and concentration of -COOH groups on the coating's surface. Optimized coatings have shown high sensitivity towards dengue Ab molecules, revealing fundamental aspect of polymer-protein interfaces as a function of surface -COOH groups' concentration.

Multidrug transport protein norM from vibrio cholerae simultaneously couples to sodium- and proton-motive force.

PubMed

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W

2014-05-23

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na(+) or H(+). We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na(+) or H(+) binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Rate Dependence in Force Networks of Sheared Granular Materials

NASA Astrophysics Data System (ADS)

Hartley, Robert; Behringer, Robert P.

2003-03-01

We describe experiments that explore rate dependence in force networks of dense granular materials undergoing slow deformation by shear and by compression. The experiments were carried out using 2D photoelastic particles so that it was possible to visualize forces at the grain scale. Shear experiments were carried out in a Couette geometry with a rate Ω. Compression experiments were carried out by repetitive compaction via a piston in a rigid chamber at comparable rates to the shear experiments. Under shearing the mean stress/force grew logarithmically with Ω for at least four decades. For compression, no dependence of the mean stress on rate was observed. In related measurements, we observed relaxation of stress in static samples that had been sheared and where the shearing was abruptly stopped. Relaxation of the force network occured over time scales of days. No relaxation of the force network was observable for uniformly compressed static samples. These results are of particular interest because they provide insight into creep and failure in granular materials.

Effect of the centrifugal force on domain chaos in Rayleigh-Bénard convection.

PubMed

Becker, Nathan; Scheel, J D; Cross, M C; Ahlers, Guenter

2006-06-01

Experiments and simulations from a variety of sample sizes indicated that the centrifugal force significantly affects the domain-chaos state observed in rotating Rayleigh-Bénard convection-patterns. In a large-aspect-ratio sample, we observed a hybrid state consisting of domain chaos close to the sample center, surrounded by an annulus of nearly stationary nearly radial rolls populated by occasional defects reminiscent of undulation chaos. Although the Coriolis force is responsible for domain chaos, by comparing experiment and simulation we show that the centrifugal force is responsible for the radial rolls. Furthermore, simulations of the Boussinesq equations for smaller aspect ratios neglecting the centrifugal force yielded a domain precession-frequency f approximately epsilon(mu) with mu approximately equal to 1 as predicted by the amplitude-equation model for domain chaos, but contradicted by previous experiment. Additionally the simulations gave a domain size that was larger than in the experiment. When the centrifugal force was included in the simulation, mu and the domain size were consistent with experiment.

The Interaction of Streptococcal Enolase with Canine Plasminogen: The Role of Surfaces in Complex Formation

PubMed Central

Balhara, Vinod; Deshmukh, Sasmit S.; Kálmán, László; Kornblatt, Jack A.

2014-01-01

The enolase from Streptococcus pyogenes (Str enolase F137L/E363G) is a homo-octamer shaped like a donut. Plasminogen (Pgn) is a monomeric protein composed of seven discrete separated domains organized into a lock washer. The enolase is known to bind Pgn. In past work we searched for conditions in which the two proteins would bind to one another. The two native proteins in solution would not bind under any of the tried conditions. We found that if the structures were perturbed binding would occur. We stated that only the non-native Str enolase or Pgn would interact such that we could detect binding. We report here the results of a series of dual polarization interferometry (DPI) experiments coupled with atomic force microscopy (AFM), isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence. We show that the critical condition for forming stable complexes of the two native proteins involves Str enolase binding to a surface. Surfaces that attract Str enolase are a sufficient condition for binding Pgn. Under certain conditions, Pgn adsorbed to a surface will bind Str enolase. PMID:24520380

3Drefine: an interactive web server for efficient protein structure refinement

PubMed Central

Bhattacharya, Debswapna; Nowotny, Jackson; Cao, Renzhi; Cheng, Jianlin

2016-01-01

3Drefine is an interactive web server for consistent and computationally efficient protein structure refinement with the capability to perform web-based statistical and visual analysis. The 3Drefine refinement protocol utilizes iterative optimization of hydrogen bonding network combined with atomic-level energy minimization on the optimized model using a composite physics and knowledge-based force fields for efficient protein structure refinement. The method has been extensively evaluated on blind CASP experiments as well as on large-scale and diverse benchmark datasets and exhibits consistent improvement over the initial structure in both global and local structural quality measures. The 3Drefine web server allows for convenient protein structure refinement through a text or file input submission, email notification, provided example submission and is freely available without any registration requirement. The server also provides comprehensive analysis of submissions through various energy and statistical feedback and interactive visualization of multiple refined models through the JSmol applet that is equipped with numerous protein model analysis tools. The web server has been extensively tested and used by many users. As a result, the 3Drefine web server conveniently provides a useful tool easily accessible to the community. The 3Drefine web server has been made publicly available at the URL: http://sysbio.rnet.missouri.edu/3Drefine/. PMID:27131371

Force development and intracellular Ca2+ in intact cardiac muscles from gravin mutant mice.

PubMed

Li, Zhitao; Singh, Sonal; Suryavanshi, Santosh V; Ding, Wengang; Shen, Xiaoxu; Wijaya, Cori S; Gao, Wei Dong; McConnell, Bradley K

2017-07-15

Gravin (AKAP12) is an A-kinase-anchoring-protein that scaffolds protein kinase A (PKA), β 2 -adrenergic receptor (β 2 -AR), protein phosphatase 2B and protein kinase C. Gravin facilitates β 2 -AR-dependent signal transduction through PKA to modulate cardiac excitation-contraction coupling and its removal positively affects cardiac contraction. Trabeculae from the right ventricles of gravin mutant (gravin-t/t) mice were employed for force determination. Simultaneously, corresponding intracellular Ca 2+ transient ([Ca 2+ ] i ) were measured. Twitch force (T f )-interval relationship, [Ca 2+ ] i -interval relationship, and the rate of decay of post-extrasysolic potentiation (R f ) were also obtained. Western blot analysis were performed to correlate sarcomeric protein expression with alterations in calcium cycling between the WT and gravin-t/t hearts. Gravin-t/t muscles had similar developed force compared to WT muscles despite having lower [Ca 2+ ] i at any given external Ca 2+ concentration ([Ca 2+ ] o ). The time to peak force and peak [Ca 2+ ] i were slower and the time to 75% relaxation was significantly prolonged in gravin-t/t muscles. Both T f -interval and [Ca 2+ ] i -interval relations were depressed in gravin-t/t muscles. R f , however, did not change. Furthermore, Western blot analysis revealed decreased ryanodine receptor (RyR2) phosphorylation in gravin-t/t hearts. Gravin-t/t cardiac muscle exhibits increased force development in responsiveness to Ca 2+ . The Ca 2+ cycling across the SR appears to be unaltered in gravin-t/t muscle. Our study suggests that gravin is an important component of cardiac contraction regulation via increasing myofilament sensitivity to calcium. Further elucidation of the mechanism can provide insights to role of gravin if any in the pathophysiology of impaired contractility. Copyright © 2017 Elsevier B.V. All rights reserved.

Langmuir-Blodgett nanotemplates for protein crystallography.

PubMed

Pechkova, Eugenia; Nicolini, Claudio

2017-12-01

The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.

Dynamic light scattering for the characterization and counting of extracellular vesicles: a powerful noninvasive tool

NASA Astrophysics Data System (ADS)

Palmieri, Valentina; Lucchetti, Donatella; Gatto, Ilaria; Maiorana, Alessandro; Marcantoni, Margherita; Maulucci, Giuseppe; Papi, Massimiliano; Pola, Roberto; De Spirito, Marco; Sgambato, Alessandro

2014-09-01

Extracellular vesicles (EVs) are cell-to-cell shuttles that have recently drawn interest both as drug delivery platforms and disease biomarkers. Despite the increasingly recognized relevance of these vesicles, their detection, and characterization still have several technical drawbacks. In this paper, we accurately assess the size distribution and concentration of EVs by using a high-throughput non-perturbative technique such as Dynamic Light Scattering (DLS). The vesicle radii distribution, as further confirmed by Atomic Force Microscopy experiments, ranges from 10 to 80 nm and appears very asymmetric towards larger radii with a main peak at roughly 30 nm. By combining DLS and Bradford assay, we also demonstrate the feasibility of recovering the concentration and its distribution of proteins contained inside vesicles. The sensitivity of our approach allows to detect protein concentrations as low as 0.01 mg/ml.

Basal body stability and ciliogenesis requires the conserved component Poc1

PubMed Central

Osborn, Daniel P.S.; Giddings, Thomas H.; Beales, Philip L.; Winey, Mark

2009-01-01

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis. PMID:20008567

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

PubMed

Mandal, Soumit Sankar; Merz, Dale R; Buchsteiner, Maximilian; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

2017-06-06

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

PubMed Central

Mandal, Soumit Sankar; Buchsteiner, Maximilian; Dima, Ruxandra I.; Rief, Matthias; Žoldák, Gabriel

2017-01-01

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD’s ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements. PMID:28533394

Study of the interaction of deoxynivalenol with human serum albumin by spectroscopic technique and molecular modelling.

PubMed

Li, Yuqin; Wang, Hao; Jia, Baoxiu; Liu, Caihong; Liu, Ke; Qi, Yongxiu; Hu, Zhide

2013-01-01

The mechanism of interaction between deoxynivalenol (DON) and human serum albumin (HSA) was studied using spectroscopic methods including fluorescence spectra, UV-VIS, Fourier transform infrared (FT-IR) and circular dichroism (CD). The quenching mechanism was investigated in terms of the association constants, number of binding sites and basic thermodynamic parameters. The distance between the HSA donor and the acceptor DON was 2.80 nm as derived from fluorescence resonance energy transfer. The secondary structure compositions of free HSA and its DON complexes were estimated by the FT-IR spectra. Alteration of the secondary protein structure in the presence of DON was confirmed by UV-VIS and CD spectroscopy. Molecular modelling revealed that a DON-protein complex was stabilised by hydrophobic forces and hydrogen bonding. It was potentially useful for elucidating the toxigenicity of DON when combined with biomolecular function effect, transmembrane transport, toxicological testing and the other experiments.

Age-related Differences in Dystrophin: Impact on Force Transfer Proteins, Membrane Integrity, and Neuromuscular Junction Stability.

PubMed

Hughes, David C; Marcotte, George R; Marshall, Andrea G; West, Daniel W D; Baehr, Leslie M; Wallace, Marita A; Saleh, Perrie M; Bodine, Sue C; Baar, Keith

2017-05-01

The loss of muscle strength with age has been studied from the perspective of a decline in muscle mass and neuromuscular junction (NMJ) stability. A third potential factor is force transmission. The purpose of this study was to determine the changes in the force transfer apparatus within aging muscle and the impact on membrane integrity and NMJ stability. We measured an age-related loss of dystrophin protein that was greatest in the flexor muscles. The loss of dystrophin protein occurred despite a twofold increase in dystrophin mRNA. Importantly, this disparity could be explained by the four- to fivefold upregulation of the dystromir miR-31. To compensate for the loss of dystrophin protein, aged muscle contained increased α-sarcoglycan, syntrophin, sarcospan, laminin, β1-integrin, desmuslin, and the Z-line proteins α-actinin and desmin. In spite of the adaptive increase in other force transfer proteins, over the 48 hours following lengthening contractions, the old muscles showed more signs of impaired membrane integrity (fourfold increase in immunoglobulin G-positive fibers and 70% greater dysferlin mRNA) and NMJ instability (14- to 96-fold increases in Runx1, AchRδ, and myogenin mRNA). Overall, these data suggest that age-dependent alterations in dystrophin leave the muscle membrane and NMJ more susceptible to contraction-induced damage even before changes in muscle mass are obvious. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Representation of Ion–Protein Interactions Using the Drude Polarizable Force-Field

PubMed Central

2016-01-01

Small metal ions play critical roles in numerous biological processes. Of particular interest is how metalloenzymes are allosterically regulated by the binding of specific ions. Understanding how ion binding affects these biological processes requires atomic models that accurately treat the microscopic interactions with the protein ligands. Theoretical approaches at different levels of sophistication can contribute to a deeper understanding of these systems, although computational models must strike a balance between accuracy and efficiency in order to enable long molecular dynamics simulations. In this study, we present a systematic effort to optimize the parameters of a polarizable force field based on classical Drude oscillators to accurately represent the interactions between ions (K+, Na+, Ca2+, and Cl–) and coordinating amino-acid residues for a set of 30 biologically important proteins. By combining ab initio calculations and experimental thermodynamic data, we derive a polarizable force field that is consistent with a wide range of properties, including the geometries and interaction energies of gas-phase ion/protein-like model compound clusters, and the experimental solvation free-energies of the cations in liquids. The resulting models display significant improvements relative to the fixed-atomic-charge additive CHARMM C36 force field, particularly in their ability to reproduce the many-body electrostatic nonadditivity effects estimated from ab initio calculations. The analysis clarifies the fundamental limitations of the pairwise additivity assumption inherent in classical fixed-charge force fields, and shows its dramatic failures in the case of Ca2+ binding sites. These optimized polarizable models, amenable to computationally efficient large-scale MD simulations, set a firm foundation and offer a powerful avenue to study the roles of the ions in soluble and membrane transport proteins. PMID:25578354

Protein folding: complex potential for the driving force in a two-dimensional space of collective variables.

PubMed

Chekmarev, Sergei F

2013-10-14

Using the Helmholtz decomposition of the vector field of folding fluxes in a two-dimensional space of collective variables, a potential of the driving force for protein folding is introduced. The potential has two components. One component is responsible for the source and sink of the folding flows, which represent respectively, the unfolded states and the native state of the protein, and the other, which accounts for the flow vorticity inherently generated at the periphery of the flow field, is responsible for the canalization of the flow between the source and sink. The theoretical consideration is illustrated by calculations for a model β-hairpin protein.

Protein displacements under external forces: An atomistic Langevin dynamics approach.

PubMed

Gnandt, David; Utz, Nadine; Blumen, Alexander; Koslowski, Thorsten

2009-02-28

We present a fully atomistic Langevin dynamics approach as a method to simulate biopolymers under external forces. In the harmonic regime, this approach permits the computation of the long-term dynamics using only the eigenvalues and eigenvectors of the Hessian matrix of second derivatives. We apply this scheme to identify polymorphs of model proteins by their mechanical response fingerprint, and we relate the averaged dynamics of proteins to their biological functionality, with the ion channel gramicidin A, a phosphorylase, and neuropeptide Y as examples. In an environment akin to dilute solutions, even small proteins show relaxation times up to 50 ns. Atomically resolved Langevin dynamics computations have been performed for the stretched gramicidin A ion channel.

Direct observation of the transition from calcite to aragonite growth as induced by abalone shell proteins.

PubMed Central

Thompson, J B; Paloczi, G T; Kindt, J H; Michenfelder, M; Smith, B L; Stucky, G; Morse, D E; Hansma, P K

2000-01-01

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface. PMID:11106633

Force Field Development and Molecular Dynamics of [NiFe] Hydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Dayle MA; Xiong, Yijia; Straatsma, TP

2012-05-09

Classical molecular force-field parameters describing the structure and motion of metal clusters in [NiFe] hydrogenase enzymes can be used to compare the dynamics and thermodynamics of [NiFe] under different oxidation, protonation, and ligation circumstances. Using density functional theory (DFT) calculations of small model clusters representative of the active site and the proximal, medial, and distal Fe/S metal centers and their attached protein side chains, we have calculated classical force-field parameters for [NiFe] in reduced and oxidized states, including internal coordinates, force constants, and atom-centered charges. Derived force constants revealed that cysteinate ligands bound to the metal ions are more flexiblemore » in the Ni-B active site, which has a bridging hydroxide ligand, than in the Ni-C active site, which has a bridging hydride. Ten nanosecond all-atom, explicit-solvent MD simulations of [NiFe] hydrogenase in oxidized and reduced catalytic states established the stability of the derived force-field parameters in terms of C{alpha} and metal cluster fluctuations. Average active site structures from the protein MD simulations are consistent with [NiFe] structures from the Protein Data Bank, suggesting that the derived force-field parameters are transferrable to other hydrogenases beyond the structure used for testing. A comparison of experimental H{sub 2}-production rates demonstrated a relationship between cysteinate side chain rotation and activity, justifying the use of a fully dynamic model of [NiFe] metal cluster motion.« less

[Atomic force microscopy fishing of gp120 on immobilized aptamer and its mass spectrometry identification].

PubMed

Bukharina, N S; Ivanov, Yu D; Pleshakova, T O; Frantsuzov, P A; Andreeva, E Yu; Kaysheva, A L; Izotov, A A; Pavlova, T I; Ziborov, V S; Radko, S P; Archakov, A I

2015-01-01

A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.

Dendrimer-protein interactions versus dendrimer-based nanomedicine.

PubMed

Shcharbin, Dzmitry; Shcharbina, Natallia; Dzmitruk, Volha; Pedziwiatr-Werbicka, Elzbieta; Ionov, Maksim; Mignani, Serge; de la Mata, F Javier; Gómez, Rafael; Muñoz-Fernández, Maria Angeles; Majoral, Jean-Pierre; Bryszewska, Maria

2017-04-01

Dendrimers are hyperbranched polymers belonging to the huge class of nanomedical devices. Their wide application in biology and medicine requires understanding of the fundamental mechanisms of their interactions with biological systems. Summarizing, electrostatic force plays the predominant role in dendrimer-protein interactions, especially with charged dendrimers. Other kinds of interactions have been proven, such as H-bonding, van der Waals forces, and even hydrophobic interactions. These interactions depend on the characteristics of both participants: flexibility and surface charge of a dendrimer, rigidity of protein structure and the localization of charged amino acids at its surface. pH and ionic strength of solutions can significantly modulate interactions. Ligands and cofactors attached to a protein can also change dendrimer-protein interactions. Binding of dendrimers to a protein can change its secondary structure, conformation, intramolecular mobility and functional activity. However, this strongly depends on rigidity versus flexibility of a protein's structure. In addition, the potential applications of dendrimers to nanomedicine are reviwed related to dendrimer-protein interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrostatic Effects in Filamentous Protein Aggregation

PubMed Central

Buell, Alexander K.; Hung, Peter; Salvatella, Xavier; Welland, Mark E.; Dobson, Christopher M.; Knowles, Tuomas P.J.

2013-01-01

Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules. PMID:23473495

Protein S-sulfenylation is a fleeting molecular switch that regulates non-enzymatic oxidative folding

NASA Astrophysics Data System (ADS)

Beedle, Amy E. M.; Lynham, Steven; Garcia-Manyes, Sergi

2016-08-01

The post-translational modification S-sulfenylation functions as a key sensor of oxidative stress. Yet the dynamics of sulfenic acid in proteins remains largely elusive due to its fleeting nature. Here we use single-molecule force-clamp spectroscopy and mass spectrometry to directly capture the reactivity of an individual sulfenic acid embedded within the core of a single Ig domain of the titin protein. Our results demonstrate that sulfenic acid is a crucial short-lived intermediate that dictates the protein's fate in a conformation-dependent manner. When exposed to the solution, sulfenic acid rapidly undergoes further chemical modification, leading to irreversible protein misfolding; when cryptic in the protein's microenvironment, it readily condenses with a neighbouring thiol to create a protective disulfide bond, which assists the functional folding of the protein. This mechanism for non-enzymatic oxidative folding provides a plausible explanation for redox-modulated stiffness of proteins that are physiologically exposed to mechanical forces, such as cardiac titin.

The effects of differing response-force requirements on fixed-ratio responding of rats.

PubMed Central

Alling, K; Poling, A

1995-01-01

Rats were exposed to two-component multiple schedules of food delivery. In the first experiment, 15 responses were required to produce food in both components. A downward force of 0.25 N (25 g) was always required to operate the response lever in one component. In the other, the required force was 0.25, 0.50, 1.00, or 2.00 N (25, 50, 100, or 200 g). In the second experiment, 0.25 N of force operated the lever in one component, but in the other, the force requirement for five consecutive responses at the beginning, middle, or end of each ratio was increased from 0.25 to 2.00 N. In the third experiment, the number of responses required to produce food was reduced from 15 to 5, and then to 1. Again, the effects of altering response force from 0.25 to 2.00 N were examined. In general, as response force increased in all experiments, mean response rates decreased and mean interresponse times increased. PMID:7751836