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In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

The elastic properties of the double-stranded DNA handles used in optical tweezers experiments on biomolecules are customarily modeled by an extensible worm-like chain model. Fitting such a model to experimental data, however, is no trivial task, as the function depends on four parameters in a highly non-linear fashion. We hereby propose a method to bypass the fitting procedure and obtain an empirical force vs. extension curve that accurately reproduces the elasticity of the handles.

Into the fold: protein domains reshuffled Protein molecules often include domains that can be distinguished as relatively separate regions in their three-dimensional structure, but how such domains communicate during folding or enzymatic function is largely unclear. Shank et al . have now developed a new technology to study this using single-molecule optical tweezers acting via DNA 'handles' to pull on a protein from different directions while monitoring the energetics of unfolding and refolding events in regions away from those submitted to mechanical forces. Comparing topological variants of a protein — the two-domain protein T4 lysozyme that is a familiar model for folding studies — they then derive new rules of cooperation between sub-domains and suggest how evolution may select reshuffled gene topologies that bypass folding dead-ends. Proteins often comprise domains that can be distinguished as relatively separate regions in the three-dimensional structure. Communication between these domains is important for catalysis, regulation and folding, but how they communicate is largely unclear. Here, single-molecule optical tweezers were used to pull on a protein while monitoring the energetics of unfolding and refolding events in disparate regions. By comparing topological variations of the same protein, new rules of cooperation between domains were derived. The three-dimensional structures of proteins often show a modular architecture comprised of discrete structural regions or domains. Cooperative communication between these regions is important for catalysis, regulation and efficient folding; lack of coupling has been implicated in the formation of fibrils and other misfolding pathologies 1 . How different structural regions of a protein communicate and contribute to a protein’s overall energetics and folding, however, is still poorly understood. Here we use a single-molecule optical tweezers approach to induce the selective unfolding of particular regions of T4 lysozyme and monitor the effect on other regions not directly acted on by force. We investigate how the topological organization of a protein (the order of structural elements along the sequence) affects the coupling and folding cooperativity between its domains. To probe the status of the regions not directly subjected to force, we determine the free energy changes during mechanical unfolding using Crooks’ fluctuation theorem. We pull on topological variants (circular permutants) and find that the topological organization of the polypeptide chain critically determines the folding cooperativity between domains and thus what parts of the folding/unfolding landscape are explored. We speculate that proteins may have evolved to select certain topologies that increase coupling between regions to avoid areas of the landscape that lead to kinetic trapping and misfolding.

Imbalances of cellular proteostasis are linked to ageing and human diseases, including neurodegenerative and neuromuscular diseases. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) together form a crucial core of the molecular chaperone family that plays a vital role in maintaining cellular proteostasis by shielding client proteins against aggregation and misfolding. sHSPs are thought to act as the first line of defence against protein unfolding/misfolding and have been suggested to act as “sponges” that rapidly sequester these aberrant species for further processing, refolding, or degradation, with the assistance of the HSP70 chaperone system. Understanding how these chaperones work at the molecular level will offer unprecedented insights for their manipulation as therapeutic avenues for the treatment of ageing and human disease. The evolution in single-molecule force spectroscopy techniques, such as optical tweezers (OT) and atomic force microscopy (AFM), over the last few decades have made it possible to explore at the single-molecule level the structural dynamics of HSPs and sHSPs and to examine the key molecular mechanisms underlying their chaperone activities. In this paper, we describe the working principles of OT and AFM and the experimental strategies used to employ these techniques to study molecular chaperones. We then describe the results of some of the most relevant single-molecule manipulation studies on HSPs and sHSPs and discuss how these findings suggest a more complex physiological role for these chaperones than previously assumed.

We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H.

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external Stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world's experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26-29 August 2018.

The mechanisms underlying transthyretin‐related amyloidosis in vivo remain unclear. The abundance of the 49–127 transthyretin fragment in ex vivo deposits suggests that a proteolytic cleavage has a crucial role in destabilizing the tetramer and releasing the highly amyloidogenic 49–127 truncated protomer. Here, we investigate the mechanism of cleavage and release of the 49–127 fragment from the prototypic S52P variant, and we show that the proteolysis/fibrillogenesis pathway is common to several amyloidogenic variants of transthyretin and requires the action of biomechanical forces provided by the shear stress of physiological fluid flow. Crucially, the non‐amyloidogenic and protective T119M variant is neither cleaved nor generates fibrils under these conditions. We propose that a mechano‐enzymatic mechanism mediates transthyretin amyloid fibrillogenesis in vivo . This may be particularly important in the heart where shear stress is greatest; indeed, the 49–127 transthyretin fragment is particularly abundant in cardiac amyloid. Finally, we show that existing transthyretin stabilizers, including tafamidis, inhibit proteolysis‐mediated transthyretin fibrillogenesis with different efficiency in different variants; however, inhibition is complete only when both binding sites are occupied. Synopsis Selective proteolysis of TTR generates a highly amyloidogenic truncated protomer. Shear stress generated by turbulent flow of physiological fluids makes TTR susceptible to cleavage. This mechanism may play a crucial role in the development of cardiac TTR amyloidosis, and offers new therapeutic targets for treating the disease. Shear forces are required to prime proteolysis of wild‐type and other variant TTRs and to release the amyloidogenic fragment. These forces are present in the heart, offering an explanation for tissue specificity in cardiac TTR amyloidosis. TTR stabilizers, currently used to treat amyloidosis, can inhibit this mechanism; however, their efficacy differs for each variant. Graphical Abstract Selective proteolysis of TTR generates a highly amyloidogenic truncated protomer. Shear stress generated by turbulent flow of physiological fluids makes TTR susceptible to cleavage. This mechanism may play a crucial role in the development of cardiac TTR amyloidosis, and offers new therapeutic targets for treating the disease.

Significance Protein misfolding can lead to neurodegeneration. Yet, the mechanistic details of this deleterious phenomenon are largely unknown, as experimental portrayals of the early and reversible molecular events leading to misfolded conformations have so far remained highly limited. Here we use single-molecule optical tweezers to monitor the structural rearrangements leading to misfolded conformations of human neuronal calcium sensor-1, a protein linked to serious neurological disorders. We identified two distinct and calcium-dependent misfolding trajectories originating from an on-pathway folding intermediate. Remarkably for a calcium sensor, pathologically high calcium concentrations impede correct folding by increasing the occupation probabilities of the misfolded states. The results open ostensible links between protein misfolding and calcium dysregulation that could be important in neurodegeneration and its potential inhibition.

Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057–2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.

The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca changes in neurons and is linked to a wide range of neuronal functions such as regulation of neurotransmitter release, voltage-gated Ca channels and neuronal outgrowth. Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca -bound, Mg -bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca and Mg ) to its EF-hands. At pathologically high Ca concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties.