Force Spectroscopy Of Proteins Research Articles

Protein mechanics probed using simple molecular models

BackgroundSingle-molecule experimental techniques such as optical tweezers or atomic force microscopy are a direct probe of the mechanical unfolding/folding of individual proteins. They are also a means to investigate free energy landscapes. Protein force spectroscopy alone provides limited information; theoretical models relate measurements to thermodynamic and kinetic properties of the protein, but do not reveal atomic level information. By building a molecular model of the protein and probing its properties through numerical simulation, one can gauge the response to an external force for individual interatomic interactions and determine structures along the unfolding pathway. In combination, single-molecule force probes and molecular simulations contribute to uncover the rich behavior of proteins when subjected to mechanical force. Scope of reviewWe focus on how simplified protein models have been instrumental in showing how general properties of the free energy landscape of a protein relate to its response to mechanical perturbations. We discuss the role of simple protein models to explore the complexity of free energy landscapes and highlight important conceptual issues that more chemically accurate models with all-atom representations of proteins and solvent cannot easily address. Major conclusionsNative-centric, coarse-grained models, despite simplifications in chemical detail compared to all-atom models, can reproduce and interpret experimental results. They also highlight instances where the theoretical framework used to interpret single-molecule data is too simple. However, these simple models are not able to reproduce experimental findings where non-native contacts are involved. General significanceMechanical forces are ubiquitous in the cell and it is increasingly clear that the way a protein responds to mechanical perturbation is important.

The nanomechanics of individual proteins.

Mechanical forces regulate a large variety of cellular functionalities, encompassing e.g. motility, differentiation and muscle contractility. To adapt to the dynamic change in mechanical stress, the constitutive individual proteins need to reversibly stretch and recoil over long periods of time. Yet, the molecular mechanisms controlling the mechanical unfolding and refolding of proteins cannot be accessed by protein folding biochemistry experiments conducted in the bulk, because they cannot typically apply forces to individual proteins. The advent of single-molecule nanomechanical techniques, often combined with bespoke protein engineering strategies, has enabled monitoring the conformational dynamics of proteins under force with unprecedented length-, time- and force-resolution. This review focuses on the fundamental operational principles of the main single-molecule nanomechanical techniques, placing particular emphasis on the most common analytical approaches used to extract information directly from the experiments. The breadth of enabling applications highlights the most exciting and promising outputs from the nanomechanics field to date.

Multiplexed protein force spectroscopy reveals equilibrium protein folding dynamics and the low-force response of von Willebrand factor.

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force regulation, and function. So far, the field has relied primarily on atomic force microscope and optical tweezers assays that, while powerful, are limited in force resolution, throughput, and require feedback for constant force measurements. Here, we present a modular approach based on magnetic tweezers (MT) for highly multiplexed protein force spectroscopy. Our approach uses elastin-like polypeptide linkers for the specific attachment of proteins, requiring only short peptide tags on the protein of interest. The assay extends protein force spectroscopy into the low force (<1 pN) regime and enables parallel and ultra-stable measurements at constant forces. We present unfolding and refolding data for the small, single-domain protein ddFLN4, commonly used as a molecular fingerprint in force spectroscopy, and for the large, multidomain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. For both proteins, our measurements reveal exponential force dependencies of unfolding and refolding rates. We directly resolve the stabilization of the VWF A2 domain by Ca2+ and discover transitions in the VWF C domain stem at low forces that likely constitute the first steps of VWF's mechano-activation. Probing the force-dependent lifetime of biotin-streptavidin bonds, we find that monovalent streptavidin constructs with specific attachment geometry are significantly more force stable than commercial, multivalent streptavidin. We expect our modular approach to enable multiplexed force-spectroscopy measurements for a wide range of proteins, in particular in the physiologically relevant low-force regime.

Thermally assisted lubricity and negative work tails in sliding friction

We discuss and qualify a previously unnoticed connection between two different phenomena in the physics of nanoscale friction, general in nature and also met in experiments including sliding emu- lations in optical lattices, and protein force spectroscopy. The first is thermolubricity, designating the condition in which a dry nanosized slider can at sufficiently high temperature and low velocity exhibit very small viscous friction f ~ v despite strong corrugations that would commonly imply hard mechanical stick-slip f ~ log(v). The second, apparently unrelated phenomenon present in externally forced nanosystems, is the occurrence of negative work tails ("free lunches") in the work probabilty distribution, tails whose presence is necessary to fulfil the celebrated Jarzynski equality of non-equilibrium statistical mechanics. Here we prove analytically and demonstrate numerically in the prototypical classical overdamped one-dimensional point slider (Prandtl-Tomlinson) model that the presence or absence of thermolubricity is exactly equivalent to satisfaction or violation of the Jarzynski equality. The divide between the two regimes, satisfaction of Jarzynski with ther- molubricity, and violation of both, simply coincides with the total frictional work per cycle falling below or above kT respectively. This concept can, with due caution, be extended to more complex sliders, thus inviting crosscheck experiments, such as searching for free lunches in cold ion sliding as well as in forced protein unwinding, and alternatively checking for a thermolubric regime in dragged colloid monolayers. As an important byproduct, we derive a parameter-free formula expressing the linear velocity coefficient of frictional dissipated power in the thermolubric viscous regime, correcting previous empirically parametrized expressions.

Soft Vibrational Modes Predict Breaking Events during Force-Induced Protein Unfolding

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.

Molecular Calipers for Highly Precise and Accurate Measurements of Single-Protein Mechanics

Single-molecule atomic force spectroscopy (AFM) has evolved into a powerful technique toward elucidating conformational changes in proteins when exposed to applied force. AFM technologies that are currently available allow for precise measurements of proteins length changes during conformational transitions. However, because of systematic errors in piezo calibration as well as errors originating from fitting experimental data using a worm-like chain model of polymer elasticity, high-precision measurements of length changes do not necessarily translate into highly accurate measurements of length changes, resulting in uncertainty in obtaining structural information about protein conformational changes. Actually achieving highly precise and accurate force spectroscopy measurements remains a challenge. Here, we report a protein caliper method that eliminates systematic errors that occur during single-protein force spectroscopy measurements, and thus achieves highly precise and accurate length change measurements in protein mechanics studies. To do this, a series of loop elongation variants of the small protein GB1, which differ by 2, 5, 10, 15, and 24 amino acid residues, were engineered. Differential measurements of amino acid residue length obtained from different AFM setups result in a precise measure of the length of a single amino acid residue, which varies within different AFM setups because of systematic error between individual AFM piezoelectric calibrations. The measured length of a single amino acid residue from a given AFM setup is then used as a caliper for the given setup to eliminate systematic error, leading to highly accurate and precise measurements of the number of amino acid residues that are involved in a conformation change of a polypeptide chain. We further developed a more precise, robust, and model-free method to determine the apparent size of single amino acid residues and conformational changes of proteins. This method improves the accuracy of single protein force spectroscopy measurements, providing an accurate means of measuring force-induced protein conformational changes.

Encased Cantilevers for Ultra-Low-Noise Force Spectroscopy of Proteins and Ligand Receptor Complexes

The energy landscape of proteins folding and unfolding or of ligand-receptor binding can be elucidated by force spectroscopy. Laser tweezers has excellent force resolution (∼10 fN/sqrt(Hz)) but a low force limit, making it poorly suited for protein pulling. Atomic Force Microscopy has a high force limit but poor force resolution (∼200 fN/sqrt(Hz)) because of viscous interaction of the water with the cantilever. To reduce viscous damping we developed encased cantilevers in which the cantilever is kept dry. Immersed in the solution, surface tension prevents the liquid from entering into the encasement, and only few microns of the tip protrude to interact with the sample in solution. The length of the free cantilever within the encasement is easily controlled during fabrication, hence, ultra short cantilevers with resonance frequencies >1MHz can be fabricated (Figure1, a and b). Thanks to the low damping we achieve quality factors of >100 and minimal detectable forces of 12 fN/sqrt(Hz). The cantilevers can be used in any conventional AFM setup using beam deflection. The low force noise and high force limit make encased cantilevers ideal for force spectroscopy applications.View Large Image | View Hi-Res Image | Download PowerPoint Slide

An Intrusive Entropic Barrier Induced by Force

Single-molecule force spectroscopy has opened up new approaches to the study of protein dynamics. For example, an extended protein folding after an abrupt quench in the pulling force was shown to follow variable collapse trajectories marked by well-defined stages that departed from the expected two-state folding behavior that is commonly observed in bulk. Here, we explain these observations by developing a simple approach that models the free energy of a mechanically extended protein as a combination of an entropic elasticity term and a short-range potential representing enthalpic hydrophobic interactions. The resulting free energy of the molecule shows a force-dependent energy barrier of magnitude, ΔE = e(F - Fc)3/2, separating the collapsed state of the molecule from a force-driven extended conformation, that vanishes at a critical force Fc. By solving the Langevin equation under force quench conditions, we generate folding trajectories corresponding to the diffusional collapse of an extended polypeptide that mimics those observed experimentally. Further we apply this model to force extension conditions in order to investigate the role played by the force-induced energy barrier on the two-state hopping phenomena that has been observed in single protein molecules placed under a stretching force. Langevin dynamics across such force induced barrier readily demonstrates the hopping behavior observed for a variety of single molecules placed under force. Our model interprets AFM force-clamp data and accounts as well for force-extension and hopping observed in optical tweezers, thus unifying the field of protein force spectroscopy. Moreover, given that this barrier does not exist at zero force, extrapolating hopping trajectories to zero force could not be compared to bulk measurements.