Thorsten Hugel

The Study of Molecular Interactions by AFM Force Spectroscopy

Recent progress in atomic force microscopy (AFM) technology has allowed the measurement of inter- and intramolecular forces at the level of individual molecules. The mechanical manipulation of single polymer chains immobilized on solid substrates has become possible in solution, as they are spanned and stretched between the tip of an AFM cantilever and the substrate surface. This investigation of polymer chains far from their maximum entropy configurations has stimulated the refinement of existing polymer theories. From the measured force-distance curves quantitative information can be obtained on the elasticity of single macromolecules in solution, on conformational transitions along the chains, about the mechanical stability of chemical bonds and on secondary structures, as well as on the desorption of individual polymer molecules from solid substrates. Recent applications of AFM single molecule force spectroscopy reach from the study of dynamic processes in complex biological systems and intermolecular forces in colloidal systems to the investigation of new functional materials capable of performing energy transductions on the level of individual macromolecules. In this article, we present a detailed description of the experimental procedure, followed by an overview of the development, the success and the current challenges of this technique during the past five years, in which it has rapidly evolved from the first proof of principle to a highly active field of research.

The large conformational changes of Hsp90 are only weakly 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.

Peptide adsorption on a hydrophobic surface results from an interplay of solvation, surface, and intrapeptide forces.

The hydrophobic effect, i.e., the poor solvation of nonpolar parts of molecules, plays a key role in protein folding and more generally for molecular self-assembly and aggregation in aqueous media. The perturbation of the water structure accounts for many aspects of protein hydrophobicity. However, to what extent the dispersion interaction between molecular entities themselves contributes has remained unclear. This is so because in peptide folding interactions and structural changes occur on all length scales and make disentangling various contributions impossible. We address this issue both experimentally and theoretically by looking at the force necessary to peel a mildly hydrophobic single peptide molecule from a flat hydrophobic diamond surface in the presence of water. This setup avoids problems caused by bubble adsorption, cavitation, and slow equilibration that complicate the much-studied geometry with two macroscopic surfaces. Using atomic-force spectroscopy, we determine the mean desorption force of a single spider-silk peptide chain as F = 58 ± 8 pN, which corresponds to a desorption free energy of ≈5 kBT per amino acid. Our all-atomistic molecular dynamics simulation including explicit water correspondingly yields the desorption force F = 54 ± 15 pN. This observation demonstrates that standard nonpolarizable force fields used in classical simulations are capable of resolving the fine details of the hydrophobic attraction of peptides. The analysis of the involved energetics shows that water-structure effects and dispersive interactions give contributions of comparable magnitude that largely cancel out. It follows that the correct modeling of peptide hydrophobicity must take the intimate coupling of solvation and dispersive effects into account.

Dynamics of heat shock protein 90 C-terminal dimerization is an important part of its conformational cycle

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.

Building lamellae from blocks : The pathway followed in the formation of crystallites of syndiotactic polypropylene

AFM images of isothermally crystallized samples of syndiotactic polypropylene and poly(propene-co-octene) show that the lamellar crystallites are composed of blocks. Upon heating the granular structure vanishes. Observations support the view recently established by SAXS experiments that polymer crystallization is a two-step process, with the block formation near the growth front as a first and their fusion into a lamella as a second step. It is proposed that the blocks have the minimum size necessary to remain intrinsically stable. This could explain the observed invariability of the crystal thickness with changing co-unit content.

Steps in the formation of the partially crystalline state

Time- and temperature-dependent SAXS studies carried out on s-polypropylene, various s-poly(propene-co-octene)s, two poly(ethylene-co-octene)s and poly(-caprolactone) indicate that the transition from the entangled melt to the partially crystalline state occurs generally in two steps. At first, an initial form of lower order builds up which then becomes stabilized to end in the final state with lamellar morphology. AFM observations suggest that the initial structure is composed of crystal blocks in planar assemblies, which then fuse into a homogeneous lamella. The edge length of the blocks in chain direction determines also the lamellar thickness. The size of the blocks corresponds to the minimum necessary to be stable. The crystallinities after isothermal crystallization processes remain invariant over larger temperature ranges, thus demonstrating that the potential of the entangled melt to form crystals is a well-defined property.

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.

On the Relationship between Peptide Adsorption Resistance and Surface Contact Angle: A Combined Experimental and Simulation Single-Molecule Study

The force-induced desorption of single peptide chains from mixed OH/CH(3)-terminated self-assembled monolayers is studied in closely matched molecular dynamics simulations and atomic force microscopy experiments with the goal to gain microscopic understanding of the transition between peptide adsorption and adsorption resistance as the surface contact angle is varied. In both simulations and experiments, the surfaces become adsorption resistant against hydrophilic as well as hydrophobic peptides when their contact angle decreases below θ ≈ 50°-60°, thus confirming the so-called Berg limit established in the context of protein and cell adsorption. Entropy/enthalpy decomposition of the simulation results reveals that the key discriminator between the adsorption of different residues on a hydrophobic monolayer is of entropic nature and thus is suggested to be linked to the hydrophobic effect. By pushing a polyalanine peptide onto a polar surface, simulations reveal that the peptide adsorption resistance is caused by the strongly bound water hydration layer and characterized by the simultaneous gain of both total entropy in the system and total number of hydrogen bonds between water, peptide, and surface. This mechanistic insight into peptide adsorption resistance might help to refine design principles for anti-fouling surfaces.

Single molecule force measurements delineate salt, pH and surface effects on biopolymer adhesion

In this paper we probe the influence of surface properties, pH and salt on the adhesion of recombinant spider silk proteins onto solid substrates with single molecule force spectroscopy. A single engineered spider silk protein (monomeric C(16) or dimeric (QAQ)(8)NR3) is covalently bound with one end to an AFM tip, which assures long-time measurements for hours with one and the same protein. The tip with the protein is brought into contact with various substrates at various buffer conditions and then retracted to desorb the protein. We observe a linear dependence of the adhesion force on the concentration of three selected salts (NaCl, NaH(2)PO(4) and NaI) and a Hofmeister series both for anions and cations. As expected, the more hydrophobic C(16) shows a higher adhesion force than (QAQ)(8)NR3, and the adhesion force rises with the hydrophobicity of the substrate. Unexpected is the magnitude of the dependences--we never observe a change of more than 30%, suggesting a surprisingly well-regulated balance between dispersive forces, water-structure-induced forces as well as co-solute-induced forces in biopolymer adhesion.

From a ratchet mechanism to random fluctuations evolution of Hsp90's mechanochemical cycle.

The 90-kDa heat shock proteins [heat shock protein 90 (Hsp90)] are a highly conserved ATP-dependent protein family, which can be found from prokaryotic to eukaryotic organisms. In general, Hsp90s are elongated dimers with N- and C-terminal dimerization sites. In a series of publications, we have recently shown that no successive mechanochemical cycle exists for yeast Hsp90 (yHsp90) in the absence of clients or cochaperones. Here, we resolve the mechanochemical cycle of the bacterial homologue HtpG by means of two- and three-color single-molecule FRET (Förster resonance energy transfer). Unlike yHsp90, the N-terminal dynamics of HtpG is strongly influenced by nucleotide binding and turnover-its reaction cycle is driven by a mechanical ratchet mechanism. However, the C-terminal dimerization site is mainly closed and not influenced by nucleotides. The direct comparison of both proteins shows that the Hsp90 machinery has developed to a more flexible and less nucleotide-controlled system during evolution.