Grant: Discovery Grants Program - Individual

Our research aims to elucidate how proteins fold, how they interact with other molecules, and why certain conditions lead to misfolding and disease. Much of this work relies on the use of electrospray mass spectrometry (ESI-MS). ""Time-resolved"" ESI-MS, a technique developed in our laboratory, allows changes in protein compactness during folding to be monitored on a time-scale of milliseconds to seconds. Simultaneously, the overall level of hydrogen bonding, as well as interactions with noncovalent binding partners (such as other proteins) can be probed. One of our goals is to characterize the structure of short-lived folding intermediates at a previously unattainable level, using a variety of strategies. A particularly promising approach is the use of electron capture dissociation for the fragmentation of proteins in the gas phase, following pulsed hydrogen-exchange in solution. By measuring the exchange levels of individual fragments it will become possible to elucidate the temporal order of hydrogen bond formation in a spatially-resolved manner. The use of hydrodynamic focusing will provide dramatic improvements in time resolution down to around one millisecond, such that even very early folding steps can be detected. In parallel, we will employ photochemical methods for the microsecond covalent labeling of proteins. The information obtained in this way (surface exposure of side chains) will be complementary to that obtained from isotope exchange experiments (hydrogen bonding). Ultimately, these activities will allow the development of ""molecular movies"", depicting the complete sequence of conformational changes during folding. In related work, rapid hydrogen exchange will be used to investigate the link between enzyme conformational dynamics and activity. These experiments will provide an answer to the long-standing question if the internal motions of enzymes are enhanced during turnover, or whether catalysis occurs within the limitations given by the intrinsic dynamics of the resting state. Computer simulations will shed light on the exact mechanism by which protein ions are released into the gas phase during ESI. All of these activities will provide excellent opportunities for HQP training.