Protein folding, the process by which a protein assumes its three-dimensional shape, is one of the basic unsolved problems of biophysical and biochemical research. Many of the structural changes taking place during protein folding, especially during the early stages, are as yet poorly understood. This is because high-resolution structural techniques generally lack the time resolution necessary for observation of folding dynamics, whereas methods that have the required time resolution generally lack structural specificity. We propose an experimental approach that combines the structure-sensitivity of multi-dimensional NMR with the ultrafast time resolution of optical techniques. To do this, we use two-dimensional optical spectroscopy (in particular, two-dimensional optical spectroscopy and time-resolved vibrational circular dichroism) in combination with site-specific labeling of proteins. This will make it possible to obtain a structurally and temporally resolved picture of protein folding, which can be regarded as a 'molecular movie' of the folding process. With the proposed method, we will investigate structural changes during protein folding at increasing levels of complexity: from the dynamics of alpha-helix nucleation, to the formation and structural characteristics of intermediate states in small globular proteins and complex beta-sheet topologies, to the nature of biologically functional, short-lived unfolded states in signalling proteins. At each of these levels of complexity, the proposed method will be used to unravel the mechanisms behind the respective folding events.
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