Mechanical Methods for Investigating Proteins
The large number of relatively weak interactions that stabilize protein structure and protein complexes at the same time allows for the unique flexibility and structural adaptability of the complexes. Most often protein function is brought about by conformational changes that are caused by the binding of a small ligand or other macromolecule. The energy released during that binding leads to mechanical displacement and the generation of forces and mechanical work. While these concepts of cellular functions, driven by mechanical elements that are powered by chemical energy in highly efficient reactions might at first be surprising, research in the last decade has shown that this is in fact the case. Processes such as replication, transcription, translation, protein folding and unfolding, protein import, splicing, organelle transport and many more are indeed mechanical processes. Although mechanical experiments with single proteins have so far been mastered by only a few groups in the world, this technique has already revolutionized functional studies of molecular machines. Beyond ultra-sensitive analytics of the relevant conformational changes application of mechanical forces offers the unique possibility to precisely influence the conformational kinetics and thus control protein conformations. This allows the controlled population and detection of conformations that are transient and hence not detectable in the absence of force. We believe that results in this area can stimulate research in the areas B, C, D and F. Furthermore, dynamic information can be obtained in systems that are in thermodynamic equilibrium. Hence, no perturbation to the system is necessary for measuring kinetics. The available techniques (AFM, optical traps) offer an accessible force window covering the whole range from sub-piconewton (thermal forces) up to the rupture of covalent bonds at several nanonewtons (nN).
Folding and unfolding of proteins (also under investigation in the areas B and F within the Cluster) will be investigated in single molecule mechanical experiments. Special empasis will be given to the identification of folding barriers in the structurally unresolved G-protein coupled receptors (Gaub/Oesterhelt). For understanding protein folding, changing the sites of force application in single molecule experiments offers the unique possibility to control the pathway in the high dimensional energy landscape. The spatial anisotropy of folding barriers will be investigated (Rief). Unraveling protein function requires understanding their complex multiple interactions in a supra-molecular context. This will be the rearch field of the new W2 tenure track professorship “Supra-molecular biophysics of proteins” at TUM. One important question concerns the action of single chaperone units in protein folding in collaboration with Buchner and Hartl (Research Area B). Further investigations on protein folding will be pursued using optical techniques as discussed in the optics part of this section.
Molecular Motors and Transporters
Understanding the coupling between the chemistry of nucleotide hydrolysis and mechanical motion is key for understanding the function of molecular motors and transporters and hence of great importance for protein transport in living systems (area B). Mechanical in combination with optical single molecule methods are ideally suited to tackle this question. A significant fraction of the resources will be dedicated to this problem. Projects will span the whole range from classical motor enzymes to transcription enzymes and protein import machines. Schliwa/Rief will correlate the kinetic and mechanical diversity of unconventional Myosins and Kinesins with their adaptation to diverse transport processes in the cell using AFM and optical traps. Michaelis/Bräuchle will study key elements of transcription, such as RNA polymerase, initiation factors, chromatin remodeling factors, as well as DNA repair enzymes using mechanical methods. These studies will occur in close collaboration with various biological groups in CIPSM: Cramer (Area C), Hopfner (Area C), Carell (Area E) and Becker (Area D). An important challenge in the field is to elucidate how protein function is regulated through transient changes such as rapid binding events and fast conformational changes. To this end, we plan to establish a new W2 tenure track position “Protein Mechanics and Optics” at the LMU campus in Grosshadern with the aim to integrate current single-molecule approaches. For example, mechanical processes leading to modulations of the transcription elongation of RNA polymerase II could be related to transient interactions of elongation factors or conformational changes of the complex observed simultaneously with optical methods. Optical tweezers and single-molecule fluorescence lend themselves to such combined investigations.
The protein import machinery of mitochondria and chloroplasts is an impressive example for a supramolecular assembly with complex mechanical function. The Neupert and Soll groups are world leading research groups that have taken the biochemistry of these machineries to a level that now allows for the development of in vitro single molecule assays. In collaborations with Neupert, AFM (Gaub) and optical tweezers will be used to study the mechanics of mitochondrial protein import. These experiments will be able to distinguish thermal ratchet models from power-stroke models. Hugel/Soll/Schleiff will tackle chloroplast import using single molecule assays.
Mechanical Single molecule studies have so far been mainly limited to motor proteins like Myosins, Kinesins, DNA-motors etc.. Even though most enzymes cannot be classified as motors or transporters they nevertheless are mechanical machines where conformational changes are coupled to chemical reactions. That’s why mechanical single molecule experiments will be able to contribute to the understanding of the chemo-mechanical behavior of a much broader class of enzymes. The Gaub group plans to optically read the mechanical response of a single lipase enzyme (CalB). The enzymatic activity is modulated mechanically and monitored via a fluorescent product. The goal is to improve our understanding of alosteric enzyme regulation with the scope to develop strategies for switchable enzymes and ultimatively to design regulated enzyme cascades. The Rief group will employ cysteine engineering to contact an enzyme mechanically via protein or DNA spacers. This project will extend a current collaboration on enzyme mechanics of dihydrofolate reductase with the Neupert group.