Moritz Mickler

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 alpha-helix, unfolding proceeds along two distinct pathways. In the dominant pathway, unfolding starts from the detachment of the primary N-terminal beta-strand, while in the minor pathway rupture of the last, C-terminal beta-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.

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.

Abstract 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 ϕ 29 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.

Abstract An biologischen molekularen Motoren können heute Einzelmolekül‐Experimente unter physiologischen Bedingungen durchgeführt werden. Das Beispiel des DNS‐packenden Motors des Bakteriophagen Φ29 zeigt, wie sich damit Einblicke in ihre Funktionweise gewinnen lassen. Dieser Motor wandelt chemische Energie in mechanische Arbeit um. Damit schiebt er die vom Wirtsbakterium vervielfältigte virale DNS in die Proteinkapsel des neuen Bakteriophagen. Die Imitation solcher komplexer molekularer Motoren liegt noch in weiter Ferne. Das Beispiel des Azobenzol‐Polymers zeigt jedoch, wie ein synthetischer molekularer Motor funktionieren kann. Es lässt sich mit Lichtpulsen zwischen einem langen und einem kurzen Zustand hin und her schalten. Der Hebelarm eines Rasterkraft‐Mikroskops kann das wie ein Kolben in mechanische Bewegung übersetzen. Das optomechanische Verhalten dieses molekularen Motors ist bereits gut verstanden.