Explore the vast range of titles available.

In a living cell, protein function is regulated in several ways, including post-translational modifications (PTMs), protein-protein interaction, or by the global environment (e.g. crowding or phase separation). While site-specific PTMs act very locally on the protein, specific protein interactions typically affect larger (sub-)domains, and global changes affect the whole protein non-specifically. Herein, we directly observe protein regulation under three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational steady states, kinetics and protein function. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We disentangle specific and non-specific effects that control Hsp90’s ATPase function, which has remained a puzzle up to now. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results demonstrate how cellular protein regulation works by fine-tuning the conformational state space of proteins. Proteins play a wide variety of roles in the cell and interact with many other molecules. The behavior of proteins depends on their structure; yet, proteins are often flexible and will change shape, much like a tree in the wind. Nevertheless, for some of the activities that it performs, a protein must adopt one specific shape. Therefore, the likelihood that the protein will take on this specific shape directly determines how efficiently that protein can perform a specific job. The shape of a protein can be regulated by changes at several levels; these could include modifying one of the amino acid building blocks that make up that protein, binding to another protein, or by placing the protein in a part of the cell that is crowded with other large molecules. Schmid and Hugel wanted to understand how these three different types of regulation affect the structure of a protein and how they relate to its activities. The protein Hsp90 was used as a test case. It typically exists with two copies of the protein bound together, either in a parallel or a V-shape. Hsp90 plays several important roles in metabolism and can break down molecules of ATP, the so-called energy currency of the cell. All three types of regulation favored the Hsp90 pairs taking the parallel structure and increased its breakdown of ATP. The results suggest that the Hsp90 pair has a flexible structure, and that reducing this flexibility can improve Hsp90’s efficiency in carrying out its role. It was particularly unexpected that the large-scale, unspecific effect of placing the protein in a crowded environment could have such similar results to a small-scale, precise change of a single amino acid within the protein. While all three forms of regulation help to stabilize the parallel structure for Hsp90, they do this through different mechanisms, which influence the speed and the way that the protein transitions between the two structures. Schmid and Hugel believe that these results offer a new perspective on how diversely the shape and function of proteins is controlled at the molecular level, which could have wider implications for medical diagnostics and treatment.

The molecular chaperone and heat shock protein Hsp90 is part of many protein complexes in eukaryotic cells. Together with its cochaperones, Hsp90 is responsible for the maturation of hundreds of clients. Although having been investigated for decades, it still is largely unknown which components are necessary for a functional complex and how the energy of ATP hydrolysis is used to enable cyclic operation. Here we use single-molecule FRET to show how cochaperones introduce directionality into Hsp90’s conformational changes during its interaction with the client kinase Ste11. Three cochaperones are needed to couple ATP turnover to these conformational changes. All three are therefore essential for a functional cyclic operation, which requires coupling to an energy source. Finally, our findings show how the formation of sub-complexes in equilibrium followed by a directed selection of the functional complex can be the most energy efficient pathway for kinase maturation. The precise role of cochaperones and ATP hydrolysis in driving Hsp90’s chaperone cycle is largely unclear. Here, the authors use single-molecule FRET to show that several cochaperones are necessary to establish directionality in Hsp90’s conformational cycle.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology. An international blind study confirms that smFRET measurements on dynamic proteins are highly reproducible across instruments, analysis procedures and timescales, further highlighting the promise of smFRET for dynamic structural biology.

An interface or surface may be considered as a planar perturbation reflected by changes in molecular properties in the direction perpendicular to the interface or surface. As a consequence, predicted by theory and shown by experiments, crystals are often covered by a thin liquid layer of their own melt. Such crystal–melt coexistence can be related to phenomena of surface premelting, secondary nucleation and melting point depression, particularly important for small systems. Here, we employed intermittent-contact mode atomic force microscopy imaging on nanoscopic semi-cylindrical filaments of polyethylene on a substrate to observe that these filaments contained a crystalline core bounded by molten regions of rather uniform width, W soft = (9 ± 2) nm at room temperature, which increased reversibly with temperature T . Filaments smaller than ca. 2 ⋅ W soft T were completely molten. The values of W soft T compared favorably with theoretically predicted characteristic length scales in the context of nucleation, surface premelting and the melting point depression of finite size crystals. Altogether, we propose that these three phenomena are related and dominated by the intermolecular forces acting at crystal surfaces. Crystals are often covered by a thin liquid layer of their own melt which can be related to phenomena of surface premelting, secondary nucleation and melting point depression. Here the authors show that semi cylindrical filaments of polyethylene contain a crystalline core bounded by molten regions and propose that these three phenomena are related and dominated by the intermolecular forces acting at crystal surfaces.

Our current understanding of biomolecular condensate formation is largely based on observing the final near-equilibrium condensate state. Despite expectations from classical nucleation theory, pre-critical protein clusters were recently shown to form under subsaturation conditions in vitro; if similar long-lived clusters comprising more than a few molecules are also present in cells, our understanding of the physical basis of biological phase separation may fundamentally change. Here, we combine fluorescence microscopy with photobleaching analysis to quantify the formation of clusters of NELF proteins in living, stressed cells. We categorise small and large clusters based on their dynamics and their response to p38 kinase inhibition. We find a broad distribution of pre-condensate cluster sizes and show that NELF protein cluster formation can be explained as non-classical nucleation with a surprisingly flat free-energy landscape for a wide range of sizes and an inhibition of condensation in unstressed cells. The nucleation of biomolecular condensates is seldom quantified in living cells. Here, the authors show how protein clusters form before microscopically visible condensation and find a flat free-energy profile with active blocking of cluster growth.

Combining single-molecule Förster resonance energy transfer (FRET) and fluorescence lifetime information inside an anti-Brownian electrokinetic (ABEL) trap makes it possible to distinguish dozens of biomolecules in a sample mixture. This method enables extensive barcoding of biomolecules with a minimal set of chemical components and opens up a path toward biomolecule quantification in complex mixtures.

The Hsp90 chaperone is responsible for the stabilization of a large variety of regulatory proteins. Single-molecule FRET was used to examine the conformational dynamics of Hsp90 in its different nucleotide-bound states. The findings suggest that, in the absence of substrate and cochaperone proteins, Hsp90's conformational changes are not strongly coupled to ATP hydrolysis. The molecular chaperone heat-shock protein 90 (Hsp90) is one of the most abundant proteins in unstressed eukaryotic cells. Its function is dependent on an exceptionally slow ATPase reaction that involves large conformational changes. To observe these conformational changes and to understand their interplay with the ATPase function, we developed a single-molecule assay that allows examination of yeast Hsp90 dimers in real time under various nucleotide conditions. We detected conformational fluctuations between open and closed states on timescales much faster than the rate of ATP hydrolysis. The compiled distributions of dwell times allow us to assign all rate constants to a minimal kinetic model for the conformational changes of Hsp90 and to delineate the influence of ATP hydrolysis. Unexpectedly, in this model ATP lowers two energy barriers almost symmetrically, such that little directionality is introduced. Instead, stochastic, thermal fluctuations of Hsp90 are the dominating processes.

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We test them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models. The ability to infer quantitative kinetic information from single-molecule FRET (smFRET) data can be challenging. Here the authors perform a blind benchmark study assessing different analysis tools used to infer kinetic rate constants from smFRET trajectories, testing on simulated and experimental data.

Time series of conformational dynamics in proteins are usually evaluated with hidden Markov models (HMMs). This approach works well if the number of states and their connectivity is known. However, for the multi-domain protein Hsp90, a standard HMM analysis with optimization of the BIC (Bayesian information criterion) cannot explain long-lived states well. Therefore, here we employ constrained HMMs, which neglect transitions between states by including assumptions. Gradually tuning a model with justified and focused changes allows us to improve its effectiveness and the score of the BIC. This became possible by analyzing time traces with several thousand observable transitions and, therefore, superb statistics. In this scheme, we also monitor the residences in the states reconstructed by the model, aiming to find exponentially distributed dwell times. We show how introducing new states can achieve these statistics but also point out limitations, e.g. for substantial similarity of two states connected to a common neighbor. One of the states displays the lowest free energy and could be the idle open ‘waiting state’, in which Hsp90 waits for the binding of nucleotides, cochaperones, or clients.

Coupling of ribosomal translation with cotranslational protein folding is essential for cellular homeostasis. In eukaryotes, Hsp70 and its J-domain cochaperone, the heterodimeric ribosome-associated complex (RAC), are central to this process; however, mechanistic insights into the coordination of Hsp70 function with translation remain limited. Here, we present two cryo-EM structures of the ribosome-bound yeast Hsp70 Ssb, identifying Rpl25/uL23 as the ribosomal binding site and revealing its interaction with a model nascent chain. Together with detailed biochemical and mutational analyses, these structures enable us to delineate the intricate RAC-dependent cycle, which positions the substrate binding domain of Ssb-ATP close to the tunnel exit to receive nascent chains. This arrangement allows Ssb to undergo substantial conformational changes upon ATP hydrolysis without steric clashes with the ribosome, while the substrate binding domain of Ssb, now anchored by the tightly bound nascent chain, remains close to the tunnel exit. Ribosomal translation is coupled to cotranslational protein folding, process assisted by dedicated chaperones. Here, authors present structures of the ribosome-bound yeast Hsp70 chaperone Ssb, identifying its ribosomal binding site and revealing its interactions with a model nascent chain.