Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales.

The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here, we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in molecular biology, surface chemistry and data analysis.

Enhanced statistical sampling reveals microscopic complexity in the talin mechanosensor folding energy landscape.

Statistical mechanics can describe the major conformational ensembles determining the equilibrium free-energy landscape of a folding protein. The challenge is to capture the full repertoire of low-occurrence conformations separated by high kinetic barriers that define complex landscapes. Computationally, enhanced sampling methods accelerate the exploration of molecular rare events. However, accessing the entire protein's conformational space in equilibrium experiments requires technological developments to enable extended observation times. We developed single-molecule magnetic tweezers to capture over a million individual transitions as a single talin protein unfolds and refolds under force in equilibrium. When observed at classically-probed timescales, talin folds in an apparently uncomplicated two-state manner. As the sampling time extends from minutes to days, the underlying energy landscape exhibits gradually larger signatures of complexity, involving a finite number of well-defined rare conformations. A fluctuation analysis allows us to propose plausible structures of each low-probability conformational state. The physiological relevance of each distinct conformation can be connected to the binding of the cytoskeletal protein vinculin, suggesting an extra layer of complexity in talin-mediated mechanotransduction. More generally, our experiments directly test the fundamental notion that equilibrium dynamics depend on the observation timescale.

A Single-Molecule Strategy to Capture Non-native Intramolecular and Intermolecular Protein Disulfide Bridges.

Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the same protein (intramolecular) or with a neighboring protein (intermolecular), frequently due to changes in the cellular redox potential. The reversible formation of non-native disulfides is intimately linked to alterations in protein function; while they can provide a mechanism to protect against cysteine overoxidation, they are also involved in the early stages of protein multimerization, a hallmark of several protein aggregation diseases. Yet their identification using current protein chemistry technology remains challenging, mainly because of their fleeting reactivity. Here, we use single-molecule spectroscopy AFM and molecular dynamics simulations to capture both intra- and intermolecular disulfide bonds in γD-crystallin, a cysteine-rich, structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as a conformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution.

Molecular Fluctuations as a Ruler of Force-Induced Protein Conformations.

Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis examines protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein's end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically stable states scales with the protein's contour length increment, in agreement with the sequence-independent freely jointed chain model of polymer physics. Our findings will help elucidate the conformational dynamics of proteins exposed to mechanical force at high resolution which are of central importance in mechanosensing and mechanotransduction.

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.

Forcing the reversibility of a mechanochemical reaction.

Mechanical force modifies the free-energy surface of chemical reactions, often enabling thermodynamically unfavoured reaction pathways. Most of our molecular understanding of force-induced reactivity is restricted to the irreversible homolytic scission of covalent bonds and ring-opening in polymer mechanophores. Whether mechanical force can by-pass thermodynamically locked reactivity in heterolytic bimolecular reactions and how this impacts the reaction reversibility remains poorly understood. Using single-molecule force-clamp spectroscopy, here we show that mechanical force promotes the thermodynamically disfavored SN2 cleavage of an individual protein disulfide bond by poor nucleophilic organic thiols. Upon force removal, the transition from the resulting high-energy unstable mixed disulfide product back to the initial, low-energy disulfide bond reactant becomes suddenly spontaneous, rendering the reaction fully reversible. By rationally varying the nucleophilicity of a series of small thiols, we demonstrate how force-regulated chemical kinetics can be finely coupled with thermodynamics to predict and modulate the reversibility of bimolecular mechanochemical reactions.

DNA Binding Induces a Nanomechanical Switch in the RRM1 Domain of TDP-43.

Understanding the molecular mechanisms governing protein-nucleic acid interactions is fundamental to many nuclear processes. However, how nucleic acid binding affects the conformation and dynamics of the substrate protein remains poorly understood. Here we use a combination of single molecule force spectroscopy AFM and biochemical assays to show that the binding of TG-rich ssDNA triggers a mechanical switch in the RRM1 domain of TDP-43, toggling between an entropic spring devoid of mechanical stability and a shock absorber bound-form that resists unfolding forces of ∼40 pN. The fraction of mechanically resistant proteins correlates with an increasing length of the TG n oligonucleotide, demonstrating that protein mechanical stability is a direct reporter of nucleic acid binding. Steered molecular dynamics simulations on related RNA oligonucleotides reveal that the increased mechanical stability fingerprinting the holo-form is likely to stem from a unique scenario whereby the nucleic acid acts as a "mechanical staple" that protects RRM1 from mechanical unfolding. Our approach highlights nucleic acid binding as an effective strategy to control protein nanomechanics.

Tailoring protein nanomechanics with chemical reactivity.

The nanomechanical properties of elastomeric proteins determine the elasticity of a variety of tissues. A widespread natural tactic to regulate protein extensibility lies in the presence of covalent disulfide bonds, which significantly enhance protein stiffness. The prevalent in vivo strategy to form disulfide bonds requires the presence of dedicated enzymes. Here we propose an alternative chemical route to promote non-enzymatic oxidative protein folding via disulfide isomerization based on naturally occurring small molecules. Using single-molecule force-clamp spectroscopy, supported by DFT calculations and mass spectrometry measurements, we demonstrate that subtle changes in the chemical structure of a transient mixed-disulfide intermediate adduct between a protein cysteine and an attacking low molecular-weight thiol have a dramatic effect on the protein's mechanical stability. This approach provides a general tool to rationalize the dynamics of S-thiolation and its role in modulating protein nanomechanics, offering molecular insights on how chemical reactivity regulates protein elasticity.

Principles for evidence-based drug formulary policy.

Expenditures for prescription drugs continue to increase, prompting insurers and health systems to adopt formulary or coverage policies restricting the use of more expensive drugs. Those establishing formulary policies face a complex array of claims regarding differences in efficacy, safety, treatment cost, or cost-effectiveness. We describe and illustrate 5 specific principles for applying research evidence to formulary decisions: (1) Experimental data should take precedence over models or simulations, and assumptions of such models should be carefully examined. (2) Morbidity or mortality outcomes should take precedence over surrogate or intermediate outcomes. (3) Claims for advantages of new treatments should consider the full range of alternatives rather than those selected by industry. (4) Variation in effects across individuals or subgroups argue against restrictions on first-line treatment, but only if those differences are predictable. (5) Variation in effects argues against requiring changes in ongoing treatment. We also discuss how economic incentives are likely to influence selection of research questions, especially research related to drug-gene interactions and to identifying new indications for existing drugs.