| 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. The structural changes taking place during protein folding, especially during the early stages, are as yet very 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 a new experimental approach that combines the structure-sensitivity of multi-dimensional NMR with the ultrafast time resolution of pulsed optical techniques. To do this, we use two-dimensional optical spectroscopy 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. The combined spatial and temporal resolution of the proposed method is far beyond that of existing methods, and will allow us to address research problems that have as yet been difficult to investigate. We will address research questions at increasing levels of complexity: from the dynamics of the elementary steps of folding, to the structural characteristics and formation of intermediate 'molten globule' states, 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 address fundamental questions regarding the mechanisms behind the respective folding events. |
