Determining the speed of intramolecular conformational changes in membrane proteins

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Membrane proteins are key components for many processes in the organism. Therefore, a thorough understanding of the functioning of these macromolecules is of utmost importance for the explanation of complex systems such as our central nervous system. In recent years, x-ray crystallography, EPR- and fluorescence spectroscopy have helped to solve many structure-function relationships so that the conformational changes of the proteins related to their function are better understood. Nevertheless, little is known about the actual dynamic behavior of the proteins on an atomic scale. It remains unknown in which manner the movements of the proteins occur and which laws can be used to describe them. Protein dynamics obtained from ensemble measurements using fluorescence or electrophysiological methods describe the distribution between the initial and the activated state of the protein, but provide no information about the movement itself. One question that arises is how fast the movements occur since this would offer information about the energy landscape of the transition process. For instance in voltage-gated ion channels, gating currents - caused by gating charge displacements - are measured in the millisecond range. Nevertheless from noise analysis, an upper limit of 5 microseconds for the transition time has been established. Protein folding is known to occur even faster. The aim of this study is to use fluorescence and phosphorescence lifetime measurements to determine upper and lower limits for the transition speed and investigate their sensitivity to the composition of the surrounding protein. This knowledge will enable us to predict with higher certainty the function of proteins based on their crystal structure and will help us to manipulate their function more directed in order to engineer constructs with defined characteristics.