Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.

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.

Anisotropic Friction in a Ligand-Protein Complex.

The effect of an externally applied directional force on molecular friction is so far poorly understood. Here, we study the force-driven dissociation of the ligand-protein complex biotin-streptavidin and identify anisotropic friction as a not yet described type of molecular friction. Using AFM-based stereographic single molecule force spectroscopy and targeted molecular dynamics simulations, we find that the rupture force and friction for biotin-streptavidin vary with the pulling angle. This observation holds true for friction extracted from Kramers' rate expression and by dissipation-corrected targeted molecular dynamics simulations based on Jarzynski's identity. We rule out ligand solvation and protein-internal friction as sources of the angle-dependent friction. Instead, we observe a heterogeneity in free energy barriers along an experimentally uncontrolled orientation parameter, which increases the rupture force variance and therefore the overall friction. We anticipate that anisotropic friction needs to be accounted for in a complete understanding of friction in biomolecular dynamics and anisotropic mechanical environments.

Aha1 regulates Hsp90's conformation and function in a stoichiometry-dependent way.

The heat shock protein 90 (Hsp90) is a molecular chaperone, which plays a key role in eukaryotic protein homeostasis. Co-chaperones assist Hsp90 in client maturation and in regulating essential cellular processes such as cell survival, signal transduction, gene regulation, hormone signaling, and neurodegeneration. Aha1 (activator of Hsp90 ATPase) is a unique co-chaperone known to stimulate the ATP hydrolysis of Hsp90, but the mechanism of their interaction is still unclear. In this report, we show that one or two Aha1 molecules can bind to one Hsp90 dimer and that the binding stoichiometry affects Hsp90's conformation, kinetics, ATPase activity, and stability. In particular, a coordination of two Aha1 molecules can be seen in stimulating the ATPase activity of Hsp90 and the unfolding of the middle domain, whereas the conformational equilibrium and kinetics are hardly affected by the stoichiometry of bound Aha1. Altogether, we show a regulation mechanism through the stoichiometry of Aha1 going far beyond a regulation of Hsp90's conformation.

Spontaneous and Selective Peptide Elongation in Water Driven by Aminoacyl Phosphate Esters and Phase Changes.

Nature chose phosphates to activate amino acids, where reactive intermediates and complex machinery drive the construction of polyamides. Outside of biology, the pathways and mechanisms that allow spontaneous and selective peptide elongation in aqueous abiotic systems remain unclear. Herein we work to uncover those pathways by following the systems chemistry of aminoacyl phosphate esters, synthetic counterparts of aminoacyl adenylates. The phosphate esters act as solubility tags, making hydrophobic amino acids and their oligomers soluble in water and enabling selective elongation and different pathways to emerge. Thus, oligomers up to dodecamers were synthesized in one flask and on the minute time scale, where consecutive additions activated autonomous phase changes. Depending on the pathway, the resulting phases initially carry nonpolar peptides and amphiphilic oligomers containing phosphate esters. During elongation and phosphate release, shorter oligomers dominate in solution, while the aggregated phase favors the presence of longer oligomers due to their self-assembly propensity. Furthermore we demonstrated that the solution phases can be isolated and act as a new environment for continuous elongation, by adding various phosphate esters. These findings suggest that the systems chemistry of aminoacyl phosphate esters can activate a selection mechanism for peptide bond formation by merging aqueous synthesis and self-assembly.

Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerization and interaction with ArlI, the motor ATPase of the archaellum.

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.

Inside a Shell-Organometallic Catalysis Inside Encapsulin Nanoreactors.

Compartmentalization of chemical reactions inside cells are a fundamental requirement for life. Encapsulins are self-assembling protein-based nanocompartments from the prokaryotic repertoire that present a highly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single-molecule Förster resonance energy transfer and 3D-MINFLUX analysis, we analyze fluorescently labeled encapsulins on a single-molecule basis. Furthermore, by equipping these capsules with a synthetic ruthenium catalyst via covalent attachment to a non-native host protein, we are able to perform in vitro catalysis and go on to show that engineered encapsulins can be used as hosts for transition metal catalysis inside living cells in confined space.

Multidomain structure and correlated dynamics determined by self-consistent FRET networks.

We present an approach that enables us to simultaneously access structure and dynamics of a multidomain protein in solution. Dynamic domain arrangements are experimentally determined by combining self-consistent networks of distance distributions with known domain structures. Local structural dynamics are correlated with the global arrangements by analyzing networks of time-resolved single-molecule fluorescence parameters. The strength of this hybrid approach is shown by an application to the flexible multidomain protein Hsp90. The average solution structure of Hsp90's closed state resembles the known X-ray crystal structure with Angstrom precision. The open state is represented by an ensemble of conformations with interdomain fluctuations of up to 25 Å. The data reveal a state-specific suppression of the submillisecond fluctuations by dynamic protein-protein interaction. Finally, the method enables localization and functional characterization of dynamic elements and domain interfaces.

Opposing Temperature Dependence of the Stretching Response of Single PEG and PNiPAM Polymers.

The response of switchable polymer blends and coatings to temperature variation is important for the development of high-performance materials. Although this has been well studied for bulk materials, a proper understanding at the molecular level, in particular for high stretching forces, is still lacking. Here we investigate the molecular details of the temperature-dependent elastic response of two widely used water-soluble polymers, namely, polyethylene glycol (PEG) and poly(N-isopropylacrylamide) (PNiPAM) with a combined approach using atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) experiments and molecular dynamics (MD) simulations. SMFS became possible by the covalent attachment of long and defined single polymers featuring a functional end group. Most interestingly, varying the temperature produces contrasting effects for PEG and PNiPAM. Surprising as these results might occur at first sight, they can be understood with the help of MD simulations in explicit water. We find that hydration is widely underestimated for the mechanics of macromolecules and that a polymer chain has competing energetic and entropic elastic components. We propose to use the temperature dependence to quantify the energetic behavior for high stretching forces. This fundamental understanding of temperature-dependent single polymer stretching response might lead to innovations like fast switchable polymer blends and coatings with polymer chains that act antagonistically.

AFM Imaging Suggests Receptor-Free Penetration of Lipid Bilayers by Toxins.

A crucial step of exotoxin action is the attack on the membrane. Many exotoxins show an architecture following the AB model, where a binding subunit translocates an "action" subunit across a cell membrane. Atomic force microscopy is an ideal technique to study these systems because of its ability to provide structural as well as dynamic information at the same time. We report first images of toxins Photorhabdus luminescens TcdA1 and Clostridium difficile TcdB on a supported lipid bilayer. A significant amount of toxin binds to the bilayer at neutral pH in the absence of receptors. Lack of diffusion indicates that toxin particles penetrate the membrane. This observation is supported by fluorescence recovery after photobleaching measurements. We mimic endocytosis by acidification while imaging the particles over time; however, we see no large conformational change. We therefore conclude that the toxin particles we imaged in neutral conditions had already formed a pore and speculate that there is no "pre-pore" state in our imaging conditions (i.e., in the absence of receptor).

Regulatory posttranslational modifications in hsp90 can be compensated by cochaperone aha1.

In this issue of Molecular Cell, Mollapour et al. (2011) show that phosphorylation of Hsp90 affects the ATPase function, chaperone function, and cochaperone binding in various ways. Several impacts can be compensated by overexpression of the cochaperone Aha1.

Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments.

Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of cross-domain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.

Conformational Dynamics of a Single Protein Monitored for 24 h at Video Rate.

We use plasmon rulers to follow the conformational dynamics of a single protein for up to 24 h at a video rate. The plasmon ruler consists of two gold nanospheres connected by a single protein linker. In our experiment, we follow the dynamics of the molecular chaperone heat shock protein 90 (Hsp90), which is known to show "open" and "closed" conformations. Our measurements confirm the previously known conformational dynamics with transition times in the second to minute time scale and reveals new dynamics on the time scale of minutes to hours. Plasmon rulers thus extend the observation bandwidth 3-4 orders of magnitude with respect to single-molecule fluorescence resonance energy transfer and enable the study of molecular dynamics with unprecedented precision.

Effects of Inhibitors on Hsp90's Conformational Dynamics, Cochaperone and Client Interactions.

The molecular chaperone and heat-shock protein Hsp90 has become a central target in anti-cancer therapy. Nevertheless, the effect of Hsp90 inhibition is still not understood at the molecular level, preventing a truly rational drug design. Here we report on the effect of the most prominent drug candidates, namely, radicicol, geldanamycin, derivatives of purine, and novobiocin, on Hsp90's characteristic conformational dynamics and the binding of three interaction partners. Unexpectedly, the global opening and closing transitions are hardly affected by Hsp90 inhibitors. Moreover, we find no significant changes in the binding of the cochaperones Aha1 and p23 nor of the model substrate Δ131Δ. This holds true for competitive and allosteric inhibitors. Therefore, direct inhibition mechanisms affecting only one molecular interaction are unlikely. We suggest that the inhibitory action observed in vivo is caused by a combination of subtle effects, which can be used in the search for novel Hsp90 inhibition mechanisms.

Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties.

Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings-such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

Cooperative Nucleotide Binding in Hsp90 and Its Regulation by Aha1.

The function of the molecular chaperone Hsp90 depends on large conformational changes, the rearrangement of local motifs, and the binding and hydrolysis of ATP. The size and complexity of the Hsp90 system impedes the detailed investigation of their interplay using standard methods. To overcome this limitation, we developed a three-color single-molecule FRET assay to study the interaction of Hsp90 with a fluorescently labeled reporter nucleotide in detail. It allows us to directly observe the cooperativity between the two nucleotide binding pockets in the protein dimer. Furthermore, our approach disentangles the protein conformation and the nucleotide binding state of Hsp90 and extracts the kinetics of the state transitions. Thereby, we can identify the kinetic causes mediating the cooperativity. We find that the presence of the first nucleotide prolongs the binding of the second nucleotide to Hsp90. In addition, we observe changes in the kinetics for both the open and the closed conformation of Hsp90 in dependence on the number of occupied nucleotide binding sites. Our analysis also reveals how the co-chaperone Aha1, known to accelerate Hsp90's ATPase activity, affects those transitions in a nucleotide-dependent and independent manner, thereby adding another layer of regulation to Hsp90.

The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function.

The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90's chaperone cycle. A unique linker, called charged linker (CL), connects the N- and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 kBT (2.7 kJ/mol) through binding of the CL to the N-domain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90's chaperone cycle governing client activation, cell viability, and stress tolerance.

Single-Molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium.

We present a simple and robust technique for extracting kinetic rate models and thermodynamic quantities from single-molecule time traces. Single-molecule analysis of complex kinetic sequences (SMACKS) is a maximum-likelihood approach that resolves all statistically relevant rates and also their uncertainties. This is achieved by optimizing one global kinetic model based on the complete data set while allowing for experimental variations between individual trajectories. In contrast to dwell-time analysis, which is the current standard method, SMACKS includes every experimental data point, not only dwell times. As a result, it works as well for long trajectories as for an equivalent set of short ones. In addition, the previous systematic overestimation of fast over slow rates is solved. We demonstrate the power of SMACKS on the kinetics of the multidomain protein Hsp90 measured by single-molecule Förster resonance energy transfer. Experiments in and out of equilibrium are analyzed and compared to simulations, shedding new light on the role of Hsp90's ATPase function. SMACKS resolves accurate rate models even if states cause indistinguishable signals. Thereby, it pushes the boundaries of single-molecule kinetics beyond those of current methods.

Mechanism of Reversible Peptide-Bilayer Attachment: Combined Simulation and Experimental Single-Molecule Study.

The binding of peptides and proteins to lipid membrane surfaces is of fundamental importance for many membrane-mediated cellular processes. Using closely matched molecular dynamics simulations and atomic force microscopy experiments, we study the force-induced desorption of single peptide chains from phospholipid bilayers to gain microscopic insight into the mechanism of reversible attachment. This approach allows quantification of desorption forces and decomposition of peptide-membrane interactions into energetic and entropic contributions. In both simulations and experiments, the desorption forces of peptides with charged and polar side chains are much smaller than those for hydrophobic peptides. The adsorption of charged/polar peptides to the membrane surface is disfavored by the energetic components, requires breaking of hydrogen bonds involving the peptides, and is favored only slightly by entropy. By contrast, the stronger adsorption of hydrophobic peptides is favored both by energy and by entropy and the desorption forces increase with increasing side-chain hydrophobicity. Interestingly, the calculated net adsorption free energies per residue correlate with experimental results of single residues, indicating that side-chain free energy contributions are largely additive. This observation can help in the design of peptides with tailored adsorption properties and in the estimation of membrane binding properties of peripheral membrane proteins.

Heat shock protein 90's mechanochemical cycle is dominated by thermal fluctuations.

The molecular chaperone and heat shock protein 90 (Hsp90) exists mainly as a homodimer in the cytoplasm. Each monomer has an ATPase in its N-terminal domain and undergoes large conformational changes during Hsp90's mechanochemical cycle. The three-color single-molecule assay and data analysis presented in the following allows one to observe at the same time nucleotide binding and the conformational changes in Hsp90. Surprisingly, and completely unlike the prior investigated systems, nucleotides can bind to the N-terminally open and closed state without strictly forcing the protein into a specific conformation. Both the transitions between the conformational states and the nucleotide binding/unbinding are mainly thermally driven. Furthermore, the two ATP binding sites show negative cooperativity; i.e., nucleotides do not bind independently to the two monomers. We thus reveal a picture of how nucleotide binding and conformational changes are connected in the molecular chaperone Hsp90, which has far-ranging consequences for its function and is distinct from previously investigated motor proteins.

Adsorption mechanism and valency of catechol-functionalized hyperbranched polyglycerols.

Nature often serves as a model system for developing new adhesives. In aqueous environments, mussel-inspired adhesives are promising candidates. Understanding the mechanism of the extraordinarily strong adhesive bonds of the catechol group will likely aid in the development of adhesives. With this aim, we study the adhesion of catechol-based adhesives to metal oxides on the molecular level using atomic force microscopy (AFM). The comparison of single catechols (dopamine) with multiple catechols on hyperbranched polyglycerols (hPG) at various pH and dwell times allowed us to further increase our understanding. In particular, we were able to elucidate how to achieve strong bonds of different valency. It was concluded that hyperbranched polyglycerols with added catechol end groups are promising candidates for durable surface coatings.