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.

Nanomanipulation of biomolecules by using single-molecule methods and computer simulations has made it possible to visualize the energy landscape of biomolecules and the structures that are sampled during the folding process. We use simulations and single-molecule force spectroscopy to map the complex energy landscape of GFP that is used as a marker in cell biology and biotechnology. By engineering internal disulfide bonds at selected positions in the GFP structure, mechanical unfolding routes are precisely controlled, thus allowing us to infer features of the energy landscape of the wild-type GFP. To elucidate the structures of the unfolding pathways and reveal the multiple unfolding routes, the experimental results are complemented with simulations of a self-organized polymer (SOP) model of GFP. The SOP representation of proteins, which is a coarse-grained description of biomolecules, allows us to perform forced-induced simulations at loading rates and time scales that closely match those used in atomic force microscopy experiments. By using the combined approach, we show that forced unfolding of GFP involves a bifurcation in the pathways to the stretched state. After detachment of an N-terminal ␣-helix, unfolding proceeds along two distinct pathways. In the dominant pathway, unfolding starts from the detachment of the primary N-terminal ␤-strand, while in the minor pathway rupture of the last, C-terminal ␤-strand initiates the unfolding process. The combined approach has allowed us to map the features of the complex energy landscape of GFP including a characterization of the structures, albeit at a coarse-grained level, of the three metastable intermediates.AFM experiments ͉ coarse-grained simulations ͉ cross-link mutants ͉ pathway bifurcation ͉ plasticity of energy landscape P rotein structures, which are astounding examples of selforganization in living systems, reach their folded states by navigating through a rugged energy landscape. Considerable progress has been made in understanding the folding mechanisms of small, single-domain proteins by using a combination of theory and experiments (1-5). Folding of many of these proteins can be approximately described as being two-state-like, that is, their energy landscape does not exhibit pronounced local minima corresponding to partially folded or misfolded structures. However, the folding energy landscapes of larger proteins, with complex topology, can be difficult to characterize because of the presence of multiple metastable intermediate structures (6,7). A detailed characterization of the structures of the intermediate states and the associated energetics is a challenge for the experimentalist. In part, the difficulty arises because complex proteins generally fold slowly and tend to aggregate in bulk experiments (8), a problem that can be avoided in mechanical unfolding of single proteins. Indeed, single-molecule mechanical methods have recently provided new possibilities for directly probing the energy landscapes of proteins and RNA because they can monito...

The molecular chaperone heat shock protein 90 (Hsp90) is an important and abundant protein in eukaryotic cells, essential for the activation of a large set of signal transduction and regulatory proteins. During the functional cycle, the Hsp90 dimer performs large conformational rearrangements. The transient N-terminal dimerization of Hsp90 has been extensively investigated, under the assumption that the C-terminal interface is stably dimerized. Using a fluorescence-based single molecule assay and Hsp90 dimers caged in lipid vesicles, we were able to separately observe and kinetically analyze N-and C-terminal dimerizations. Surprisingly, the C-terminal dimer opens and closes with fast kinetics. The occupancy of the unexpected C-terminal open conformation can be modulated by nucleotides bound to the N-terminal domain and by N-terminal deletion mutations, clearly showing a communication between the two terminal domains. Moreover our findings suggest that the C-and N-terminal dimerizations are anticorrelated. This changes our view on the conformational cycle of Hsp90 and shows the interaction of two dimerization domains. The crystal structure of the full-length Hsp90 from yeast bound to adenosine 5′-[β,γ-imido]triphosphate (AMP-PNP) and p23 revealed a compact homodimeric structure where both the N-and C-terminal domains are dimerized (7). Recent studies in solution with small-angle X-ray scattering, hydrogen exchange mass spectrometry (8-10) and single-molecule fluorescence (11) showed a highly dynamic and stochastic picture of Hsp90, where the system is in equilibrium among different N-terminal open and closed (compact) states. Up to now experiments have not been able to separately observe N-and C-terminal dimerization kinetics, and therefore the C-terminal domain was assumed to be closed on the time scale of experiments [motivated by the low K D value of 60 nM for yeast Hsp90 (12)]. Our single-molecule assay overcomes this experimental limitation and allows the investigation of the C-terminal dimerization interface independently in real time. Contrary to the general assumptions, fast opening and closing kinetics of the C-terminal domains can be found. Furthermore, we clearly detect an unexpected C-terminal open and an N-terminal closed state and a communication in between the C and N termini. Results Single-Molecule Fluorescence Resonance Energy Transfer (smFRET)Shows C-terminal Dimerization Kinetics. To directly monitor the dynamics of the C-terminal domain, we created a variant of yeast Hsp90 in which glutamine at position 560 in the C-terminal domain was exchanged against a cysteine (C560). The single cysteine in one monomer was labeled with the donor fluorophore Atto550, and in the second monomer with the acceptor fluorophore Atto647N. The dimers were then caged in lipid vesicles that were immobilized onto a solid substrate similar to the experiments by Cisse et al. (13) and by Rhoades et al. (14) (see Fig. 1 and SI Methods for details). Functionality of the investigated constructs was tested by an ATPase assa...

Novel single-molecule techniques allow the observation of single-molecular motors in real time under physiological conditions. This enables one to gain previously inaccessible information about the mechanics of molecular motors, especially their mechano-chemical coupling. As an example, we discuss the DNA import motor of the bacteriophage phi29 and protein import into chloroplasts. In contrast to these highly developed biological molecular motors, artificial molecular motors are still at an early stage of development. Nevertheless, they already give a wealth of information. Our review focuses on how the investigation of artificial and biological molecular motors can mutually enrich each other.