Improving Split Reporters of Protein-Protein Interactions through Orthology-Based Protein Engineering.

Protein-protein interactions (PPIs) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of splitFAST2, an improved reporter displaying higher brightness, lower self-complementation, and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, splitFAST2 is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

Enzyme-less nanopore detection of post-translational modifications within long polypeptides.

Means to analyse cellular proteins and their millions of variants at the single-molecule level would uncover substantial information previously unknown to biology. Nanopore technology, which underpins long-read DNA and RNA sequencing, holds potential for full-length proteoform identification. We use electro-osmosis in an engineered charge-selective nanopore for the non-enzymatic capture, unfolding and translocation of individual polypeptides of more than 1,200 residues. Unlabelled thioredoxin polyproteins undergo transport through the nanopore, with directional co-translocational unfolding occurring unit by unit from either the C or N terminus. Chaotropic reagents at non-denaturing concentrations accelerate the analysis. By monitoring the ionic current flowing through the nanopore, we locate post-translational modifications deep within the polypeptide chains, laying the groundwork for compiling inventories of the proteoforms in cells and tissues.

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 mechanical stability of proteins regulates their translocation rate into the cell nucleus.

The translocation of mechanosensitive transcription factors (TFs) across the nuclear envelope is a crucial step in cellular mechanotransduction. Yet the molecular mechanisms by which external mechanical cues control the nuclear shuttling dynamics of TFs through the nuclear pore complex (NPC) to activate gene expression are poorly understood. Here, we show that the nuclear import rate of myocardin-related transcription factor A (MRTFA) - a protein that regulates cytoskeletal dynamics via the activation of the TF serum response factor (SRF) - inversely correlates with the protein's nanomechanical stability and does not relate to its thermodynamic stability. Tagging MRTFA with mechanically resistant proteins results in the downregulation of SRF-mediated myosin light-chain 9 (MYL9) gene expression and subsequent slowing down of cell migration. We conclude that the mechanical unfolding of proteins regulates their nuclear translocation rate through the NPC, and highlight the role of the NPC as a selective mechanosensor able to discriminate forces as low as ~10 pN. The modulation of the mechanical stability of TFs may represent a new strategy for the control of gene expression.

The importance of advanced material selection and testing strategies.

The Success of new product development (NPD) projects can be increased by employing a strategic and integrated approach to material selection and testing. The value of this approach is described and real life examples provide some useful practical recommendations for those embarking on a NPD project.