Single-molecule Force Spectroscopy Studies Research Articles

Solvent Polarity Effects on the Mechanochemistry of Spiropyran Ring Opening.

The spiropyran mechanophore (SP) is employed as a reporter of molecular tension in a wide range of polymer matrices, but the influence of surrounding environment on the force-coupled kinetics of its ring opening has not been quantified. Here, we report single-molecule force spectroscopy studies of SP ring opening in five solvents that span normalized Reichardt solvent polarity factors (ETN) of 0.1-0.59. Individual multimechanophore polymers were activated under increasing tension at constant 300 nm s-1 displacement in an atomic force microscope. The extension results in a plateau in the force-extension curve, whose midpoint occurs at a transition force f* that corresponds to the force required to increase the rate constant of SP activation to approximately 30 s-1. More polar solvents lead to mechanochemical reactions that are easier to trigger; f* decreases across the series of solvents, from a high of 415 ± 13 pN in toluene to a low of 234 ± 9 pN in n-butanol. The trend in mechanochemical reactivity is consistent with the developing zwitterionic character on going from SP to the ring-opened merocyanine product. The force dependence of the rate constant (Δx‡) was calculated for all solvent cases and found to increase with ETN, which is interpreted to reflect a shift in the transition state to a later and more productlike position. The inferred shift in the transition state position is consistent with a double-well (two-step) reaction potential energy surface, in which the second step is rate determining, and the intermediate is more polar than the product.

Two-Molecule Force Spectroscopy on Proteins.

Many elastomeric proteins, which play important roles in a wide range of biological processes, exist as parallel/antiparallelly arranged dimers or multimers to perform their mechanobiological functions. For example, in striated muscle sarcomeres, the giant muscle protein titin exists as hexameric bundles to mediate the passive elasticity of muscles. However, it has not been possible to directly probe the mechanical properties of such parallelly arranged elastomeric proteins. And it remains unknown if the knowledge obtained from single-molecule force spectroscopy studies can be directly extrapolated to such parallelly/antiparallelly arranged systems. Here, we report the development of atomic force microscopy (AFM)-based two-molecule force spectroscopy to directly probe the mechanical properties of two elastomeric proteins that are arranged in parallel. We developed a twin-molecule approach to allow two parallelly arranged elastomeric proteins to be picked up and stretched simultaneously in an AFM experiment. Our results clearly revealed the mechanical features of such parallelly arranged elastomeric proteins during force-extension measurements and allowed for the determination of mechanical unfolding forces of proteins in such an experimental setting. Our study provides a general and robust experimental strategy to closely mimic the physiological condition of such parallel elastomeric protein multimers.

Single Molecule Force Spectroscopy Studies on Metalloproteins: Opportunities and Challenges.

Metalloproteins play important roles in a wide range of biological processes. Elucidating the mechanisms via which metalloproteins fold and constitute their metal centers is critical to the understanding of the functions and dynamics of metalloproteins. Owing to its superior force and length resolution, single-molecule force spectroscopy (SMFS) has evolved into a powerful tool to probe the unfolding and folding mechanisms of metalloproteins at the single level by forcing metalloproteins to unfold and then refold along a reaction coordinate defined by the applied stretching force. The folding of metalloproteins is complex and involves two interwound processes, the folding of the polypeptide chain and the constitution of the metal center. Experimental studies of the folding of metalloproteins are challenging. SMFS studies have allowed researchers to directly probe the folding and unfolding of metalloproteins at the single-molecule level and the effect of metal centers on the folding-unfolding energy landscape of metalloproteins. New mechanistic insights on the folding and unfolding of some metalloproteins have been obtained, demonstrating the power and unique advantages that SMFS techniques may offer. In this Perspective, using calcium-binding proteins and small iron-sulfur proteins as examples, I provide a concise overview of the information and insights that SMFS studies have provided to understand the folding and unfolding of metalloproteins. I also discuss the opportunities and challenges that are present in this fast-progressing area of research.

Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations.

A highly bioluminescent protein, NanoLuc (Nluc), has seen numerous applications in biological assays since its creation. We recently engineered a NanoLuc polyprotein that showed high bioluminescence but displayed a strong misfolding propensity after mechanical unfolding. Here, we present our single-molecule force spectroscopy (SMFS) studies by atomic force microscopy (AFM) and steered molecular dynamics (SMD) simulations on two new hybrid protein constructs comprised of Nluc and I91 titin domains, I91-I91-Nluc-I91-I91-I91-I91 (I912-Nluc-I914) and I91-Nluc-I91-Nluc-I91-Nluc-I91, to characterize the unfolding behavior of Nluc in detail and to further investigate its misfolding properties that we observed earlier for the I912-Nluc3-I912 construct. Our SMFS results confirm that Nluc's unfolding proceeds similarly in all constructs; however, Nluc's refolding differs in these constructs, and its misfolding is minimized when Nluc is monomeric or separated by I91 domains. Our simulations on monomeric Nluc, Nluc dyads, and Nluc triads pinpointed the origin of its mechanical stability and captured interesting unfolding intermediates, which we also observed experimentally.

Methods to study folding of alpha-helical membrane proteins in lipids.

How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by the lipids within the surrounding bilayer, but the majority of folding studies have focused on detergent-solubilized protein rather than protein in a lipid environment. There are different ways to study folding in lipid bilayers, and each method has its own advantages and disadvantages. This review will discuss folding methods which can be used to study alpha-helical membrane proteins in bicelles, liposomes, nanodiscs or native membranes. These folding methods include in vitro folding methods in liposomes such as denaturant unfolding studies, and single-molecule force spectroscopy studies in bicelles, liposomes and native membranes. This review will also discuss recent advances in co-translational folding studies, which use cell-free expression with liposomes or nanodiscs or are performed in vivo with native membranes.

Pulling Outward but Reacting Inward: Mechanically Induced Symmetry-Allowed Reactions of cis- and trans-Diester-Substituted Dichlorocyclopropanes

AbstractThe mechanically induced symmetry-allowed disrotatory ring openings of cis- and trans-gem-dichlorocyclopropane (gDCC) diesters are demonstrated through sonication and single-molecule force spectroscopy (SMFS) studies. In contrast to the previously reported symmetry-forbidden conrotatory ring opening of alkyl-tethered trans-gDCC, we show that the diester-tethered trans-gDCC primarily undergoes a symmetry-allowed disrotatory pathway even at the high forces (>2 nN) and short-time scales (ms or less) of sonication and SMFS experiments. The quantitative force-rate data obtained from SMFS data is consistent with computational models of transition-state geometry for the symmetry-allowed process, and activation lengths of 1.41 ± 0.02 Å and 1.08 ± 0.03 Å are inferred for the cis-gDCC diester and trans-gDCC diester, respectively. The strong mechanochemical coupling in the trans-gDCC is notable, given that the directionality of the applied force may appear initially to oppose the disrotatory motion associated with the reaction. The stereochemical perturbations of mechanical coupling created by the ester attachments reinforce the complexity that is possible in covalent polymer mechanochemistry and illustrate the breadth of reactivity outcomes that are available through judicious mechanophore design.

Combined in-situ attenuated total reflection-Fourier transform infrared spectroscopy and single molecule force studies of poly(acrylic acid) at electrolyte/oxide interfaces at acidic pH

Fundamental adsorption mechanisms of poly(acrylic acid) (PAA) electrolyte/oxide interfaces were analyzed by the combination of in-situ attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and single molecule force spectroscopy (SMFS). The approach aims at a fundamental understanding of initial states of polymer fouling in chemical microreactors. While the presented FTIR-data provide information on adsorption and desorption kinetics, SMFS studies reveal the corresponding interfacial and intermolecular forces. Silicon oxide and oxide covered FeCr alloy films with small concentrations of Ni were chosen as reference systems for relevant technical reactor components. Adsorption and desorption studies were performed in aqueous electrolytes at acidic pH to simulate the polymerisation process. Ex-situ ellipsometry and atomic force microscopy (AFM) studies of the adsorbed polymer layers as well as X-ray photoelectron spectroscopy (XPS) of the oxide surfaces complemented the analytical approach. The comparison of the in-situ ATR spectroscopic results and the SMFS data revealed higher molecular adhesion forces on FeCr-oxide covered FeCr alloy films in comparison to the SiOx terminated surfaces. The different interactions could be assigned to the specific coordination bonds formed between the carboxylic acid group and surface metal ions in the case of FeCr alloy films. AFM images showed related changes in interfacial film formation.

Pseudopeptide Amyloid Aggregation Inhibitors: In Silico, Single Molecule and Cell Viability Studies.

Neurodegeneration in Alzheimer’s disease (AD) is defined by pathology featuring amyloid-β (Aβ) deposition in the brain. Aβ monomers themselves are generally considered to be nontoxic, but misfold into β-sheets and aggregate to form neurotoxic oligomers. One suggested strategy to treat AD is to prevent the formation of toxic oligomers. The SG inhibitors are a class of pseudopeptides designed and optimized using molecular dynamics (MD) simulations for affinity to Aβ and experimentally validated for their ability to inhibit amyloid-amyloid binding using single molecule force spectroscopy (SMFS). In this work, we provide a review of our previous MD and SMFS studies of these inhibitors and present new cell viability studies that demonstrate their neuroprotective effects against Aβ(1–42) oligomers using mouse hippocampal-derived HT22 cells. Two of the tested SG inhibitors, predicted to bind Aβ in anti-parallel orientation, demonstrated neuroprotection against Aβ(1–42). A third inhibitor, predicted to bind parallel to Aβ, was not neuroprotective. Myristoylation of SG inhibitors, intended to enhance delivery across the blood-brain barrier (BBB), resulted in cytotoxicity. This is the first use of HT22 cells for the study of peptide aggregation inhibitors. Overall, this work will inform the future development of peptide aggregation inhibitors against Aβ toxicity.

Modulating mechanical stability of heterodimerization between engineered orthogonal helical domains

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats.

Generalized Langevin Equation as a Model for Barrier Crossing Dynamics in Biomolecular Folding.

Conformational memory in single-molecule dynamics has attracted recent attention and, in particular, has been invoked as a possible explanation of some of the intriguing properties of transition paths observed in single-molecule force spectroscopy (SMFS) studies. Here we study one candidate for a non-Markovian model that can account for conformational memory, the generalized Langevin equation with a friction force that depends not only on the instantaneous velocity but also on the velocities in the past. The memory in this model is determined by a time-dependent friction memory kernel. We propose a method for extracting this kernel directly from an experimental signal and illustrate its feasibility by applying it to a generalized Rouse model of a SMFS experiment, where the memory kernel is known exactly. Using the same model, we further study how memory affects various statistical properties of transition paths observed in SMFS experiments and evaluate the performance of recent approximate analytical theories of non-Markovian dynamics of barrier crossing. We argue that the same type of analysis can be applied to recent single-molecule observations of transition paths in protein and DNA folding.

Generalized Langevin Equation as a Model for Barrier Crossing Dynamics in Biomolecular Folding.

Conformational memory in single-molecule dynamics has attracted recent attention and, in particular, has been invoked as a possible explanation of some of the intriguing properties of transition paths observed in single-molecule force spectroscopy (SMFS) studies. Here we study one candidate for a non-Markovian model that can account for conformational memory, the generalized Langevin equation with a friction force that depends not only on the instantaneous velocity but also on the velocities in the past. The memory in this model is determined by a time-dependent friction memory kernel. We propose a method for extracting this kernel directly from an experimental signal and illustrate its feasibility by applying it to a generalized Rouse model of a SMFS experiment, where the memory kernel is known exactly. Using the same model, we further study how memory affects various statistical properties of transition paths observed in SMFS experiments and evaluate the performance of recent approximate analytical theories of non-Markovian dynamics of barrier crossing. We argue that the same type of analysis can be applied to recent single-molecule observations of transition paths in protein and DNA folding.

High-Precision Single-Molecule Characterization of the Folding of an HIV RNA Hairpin by Atomic Force Microscopy.

The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps. Although commercial atomic force microscopes (AFMs) are widely deployed and offer significant advantages in ease-of-use over custom-built optical traps, traditional AFM-based SMFS lacks the sensitivity and stability to characterize individual RNA molecules precisely. Here, we developed a high-precision SMFS assay to study RNA folding using a commercial AFM and applied it to characterize a small RNA hairpin from HIV that plays a key role in stimulating programmed ribosomal frameshifting. We achieved rapid data acquisition in a dynamic assay, unfolding and then refolding the same individual hairpin more than 1,100 times in 15 min. In comparison to measurements using optical traps, our AFM-based assay featured a stiffer force probe and a less compliant construct, providing a complementary measurement regime that dramatically accelerated equilibrium folding dynamics. Not only did kinetic analysis of equilibrium trajectories of the HIV RNA hairpin yield the traditional parameters used to characterize folding by SMFS (zero-force rate constants and distances to the transition state), but we also reconstructed the full 1D projection of the folding free-energy landscape comparable to state-of-the-art studies using dual-beam optical traps, a first for this RNA hairpin and AFM studies of nucleic acids in general. Looking forward, we anticipate that the ease-of-use of our high-precision assay implemented on a commercial AFM will accelerate studying folding of diverse nucleic acid structures.

Mechanical Softening of a Small Ubiquitin-Related Modifier Protein Due to Temperature Induced Flexibility at the Core.

Despite the growing interest in the thermal softening of proteins, the mechanistic details of it are far from understood. β-Grasp proteins have globular shape with compact structure and they are mechanically resilient. The β-clamp or mechanical clamp in them formed by the interactions between the terminal β-strands is generally associated with the protein mechanical resistance. Although previous studies showed that temperature can perturb the protein mechanical stability, the structural changes leading to the lowered mechanical resistance are not known. Here, we investigated the temperature dependent mechanical stability of small ubiquitin-related modifier 2 (SUMO2) using single-molecule force spectroscopy (SMFS) and the corresponding conformational changes using ensemble experiments. SMFS studies on the polyprotein of SUMO2 estimate a decrease in the spring constant of the protein from 4.50 to 1.35 N/m upon increasing the temperature from 5 to 45 °C. Interestingly, near-UV circular dichroism spectroscopy reveals a decrease in tertiary structure content while the overall secondary structure of the protein remains unchanged. Steady-state fluorescence and quenching studies on SUMO2 with a tryptophan mutation at the core (F60W) show that the nonpolar environment of the tryptophan is unchanged and the protein core is inaccessible to the bulk solvent, in the same temperature range. We attribute the thermal softening observed in atomic force microscopy (AFM) experiments to the reduction in tertiary structure of SUMO2. Our results provide evidence for the importance of the intramolecular interactions at the protein core along with the β-clamp or mechanical clamp in providing the mechanical resistance as well as in modulating the protein stiffness.

Communication: Transition-path velocity as an experimental measure of barrier crossing dynamics.

Experimental observation of transition paths-short events when the system of interest crosses the free energy barrier separating reactants from products-provides an opportunity to probe the dynamics of barrier crossing. Yet limitations in the experimental time resolution usually result in observing trajectories that are smoothed out, recross the transition state fewer times, and exhibit apparent velocities that are much lower than the instantaneous ones. Here we show that it is possible to define (and measure) an effective transition-path velocity which preserves exact information about barrier crossing dynamics in the following sense: the exact transition rate can be written in a form resembling that given by transition-state theory, with the mean thermal velocity replaced by the transition-path velocity. In addition, the transition-path velocity (i) ensures the exact local value of the unidirectional reactive flux at equilibrium and (ii) leads to the exact mean transition-path time required for the system to cross the barrier region separating reactants from products. We discuss the coordinate dependence of the transition path velocity and derive analytical expressions for it in the case of diffusive dynamics. These results can be used to discriminate among models of barrier crossing dynamics in single-molecule force spectroscopy studies.

A peptide-display protein scaffold to facilitate single molecule force studies of aggregation-prone peptides.

Protein aggregation is linked with the onset of several neurodegenerative disorders, including Parkinson's disease (PD), which is associated with the aggregation of α‐synuclein (αSyn). The structural mechanistic details of protein aggregation, including the nature of the earliest protein–protein interactions, remain elusive. In this study, we have used single molecule force spectroscopy (SMFS) to probe the first dimerization events of the central aggregation‐prone region of αSyn (residues 71–82) that may initiate aggregation. This region has been shown to be necessary for the aggregation of full length αSyn and is capable of forming amyloid fibrils in isolation. We demonstrate that the interaction of αSyn71‐82 peptides can be studied using SMFS when inserted into a loop of protein L, a mechanically strong and soluble scaffold protein that acts as a display system for SMFS studies. The corresponding fragment of the homolog protein γ‐synuclein (γSyn), which has a lower aggregation propensity, has also been studied here. The results from SMFS, together with native mass spectrometry and aggregation assays, demonstrate that the dimerization propensity of γSyn71‐82 is lower than that of αSyn71‐82, but that a mixed αSyn71‐82: γSyn71‐82 dimer forms with a similar propensity to the αSyn71‐82 homodimer, slowing amyloid formation. This work demonstrates the utility of a novel display method for SMFS studies of aggregation‐prone peptides, which would otherwise be difficult to study.

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.

Abstract 178: Peptide nanofibers: targeted therapies for glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is a malignant brain tumor with poor prognosis due to tumor heterogeneity, poor drug blood-brain barrier (BBB) permeability and targeting. GBM biopsies indicated the overexpression of G-protein coupled receptors (GPCRs) that when activated by neuropeptide agonists result in an antiproliferative effect. However, translation of these neuropeptides into novel therapies for GBM is frustrated by their short half-life (<5 minutes) and inability to cross the BBB. Here, we present a novel strategy based on the lipidization of peptide agonists which results in peptide amphiphiles able to self-assemble into nanofibers that can entrap brain impermeable drugs, possess an enhanced stability to enzymatic degradation, permeate across an all human in vitro BBB model and are able to target GBM cells resulting in a significant antiproliferative effect. Peptide amphiphiles were synthesized using solid-phase peptide synthesis and characterised using pyrene, thioflavin T, circular dichroism and transmission electron microscopy experiments. Nanofibers were loaded with brain impermeable cytotoxic drugs (e.g. paclitaxel) and their stability was studied in biological media (plasma, brain, liver and GBM cell lysates). BBB permeation was studied in an all human in vitro Transwell model. The antiproliferative effect of the nanofibers was evaluated on U-87 MG cells. Targeting of the nanofibers to the GPCR was evaluated using single-molecule force spectroscopy (SMFS). Peptide amphiphiles self-assemble into stable nanofibers at concentrations above 189 μM and are able to solubilize high amounts of paclitaxel (>1.8 mg) ensuring that the resulting nanomedicine can be clinically translatable. Both unloaded- and paclitaxel loaded-nanofibers showed superior stability compared to the parent neuropeptide in the presence of plasma, brain, liver and cell homogenates (>6-fold). Nanofibers elicited a strong antiproliferative effect (IC50: 5.06 μM) resulting in a cell cycle arrest at G2/M in a GPCR positive GBM cell line (U-87 MG). Loading paclitaxel (1nM) within the nanofibers resulted in a synergistic effect evidenced by a decrease in cell survival by 32%. Nanofibers counteracted the forskolin-induced increase of intracellular cAMP levels indicating that the GPCR is linked to the GαI protein known to mediate the antiproliferative effect on GBM. Confocal studies confirmed the internalization of the peptide while SMFS studies supported specific binding of the nanofibers to GPCR on U-87MG cell surface with equivalent binding probability to peptide agonists and increased residence time. Texas Red labelled nanofibers permeate across an in vitro BBB model enabling the permeation of paclitaxel (Papp: 8.45 x10-6 cm/s). Thus, described peptide nanofibers are a novel targeted nanomedicine for GBM therapy able to be clinically translated. In vivo proof of concept studies and pharmacokinetics are under way. Citation Format: Diana M. Leite, Rong Zhu, Eugen Barbu, Peter Hinterdorfer, Geoffrey J. Pilkington, Aikaterini Lalatsa. Peptide nanofibers: targeted therapies for glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 178. doi:10.1158/1538-7445.AM2017-178

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

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.

Technique reveals never-before-seen states as protein unfolds

An improved version of single-molecule force spectroscopy (SMFS) has enabled researchers to study the unfolding of a membrane protein with much higher time resolution and force precision than ever before, revealing a multitude of new details. Proteins must adopt specific three-dimensional shapes to work properly. If they don’t, they can malfunction or cause protein-misfolding conditions such as Alzheimer’s disease. Scientists would therefore like to better understand how proteins fold and unfold, including all the intermediate states they adopt along the way. One type of SMFS technique probes protein-folding intermediates by using an atomic force microscopy (AFM) cantilever to pull on a single protein molecule, unraveling it a little at a time. Most unfolding steps are reversible, so studying unfolding reveals a protein’s folding behavior as well. In the past, SMFS studies found two intermediate states in the unfolding of two helices in the membrane protein bacteriorhodopsin. But molecular dynamics simula...

Complex Dynamics in Single Molecule Force Spectroscopy from Simple Simulation Models

Single molecule force spectroscopy studies of protein folding dynamics have yielded unprecedented insight into the mechanisms of the folding reaction. Still, they usually allow for a very limited description of the process of interest (e.g. based on the reaction coordinate that is probed directly). Molecular simulations --even using very simplified models-- can give a much enriched view of the underlying molecular events. In a recent example, we explain the observations on a prototypical two-state folding protein, the cold shock protein, that in the AFM turns out to exhibit multiple intermediate states. Using a very simple topology based model for the protein we show that in fact this should not come as a surprise. Indeed, force can selectively stabilize different protein intermediates and act in a very different way compared to that of chemical denaturants.