Characterization of different conformational states of proteins by highpressure NMR spectroscopy

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The main goal of the proposal is to detect and characterize rare (excited) states of polypeptides and proteins by high-pressure NMR spectroscopy which probably are involved in physiological and pathophysiological polymerization/depolymerisation processes. As examples of high physiological importance, the two main polymers of the eukaryotic cytoskeleton actin and tubulin forming microfilaments and microtubules in the cell were selected. As examples for pathophysiological polymerisation/aggregation processes, the study of the two polypeptides Abeta (beta amyloid) and IAPP (islet amyloid forming polypeptide) will be continued that are involved in the development of Alzheimer disease and diabetes mellitus type II. Using high pressure NMR spectroscopy equilibria between different conformational states of these proteins will be characterized structurally and thermodynamically and related to the polymerizationdepolymerization process. In actin und tubulin filaments, the regulated fast polymerization and depolymerisation is crucial for a proper function of the cell. It will be characterized by high pressure NMR spectroscopy combined with more involved NMR methods such as CEST and relaxation dispersion spectroscopy. Abeta and IAPP form amyloid deposits when the diseases develop. The use of 15N and 13C enriched peptides will increase the information content dramatically. High pressure NMR spectroscopy will be used to delineate the free energy landscape and should give information if rare conformational states are involved in the pathological polymerisation. Their existence may open a new way for the development of intrinsic allosteric inhibitors. By pressure-jump spectroscopy, the kinetics of the depolymerisation will be studied in more detail. The characterization of the proteins will be supplemented by other biophysical methods such as high pressure fluorescence and infrared spectroscopy provided by the partners of the consortium. Since ceramic cells that withstand pressures up to 300 MPa are now commercially available, we will be able to get NMR data in a pressure range not accessible to us before. This will increase dramatically the information content of the obtained NMR data and increase the accuracy of the obtained thermodynamic parameters. In addition, the high-pressure NMR study of the conformational equilibria occurring in small pressure-activated catalytical peptides and foldamers will be continued, synthetic polypeptides that can mimic different functional states of naturally occurring polypeptides. For providing an experimental basis for the improvement of molecular dynamics calculations and quantum chemical approaches to pressure effects, the pressure response of small molecules such as TMAO will be elucidated. Here, also the possible mode of action of cosmotropic compounds will be studied in detail, especially their putative direct interactions with model peptides and proteins.