Our research group has a continuing interest in understanding the molecular mechanisms responsible for morphologic changes in mitochondria. We are focusing on specific molecular interactions that regulate these mitochondria changes following a cell death (apoptotic) initiation. We are concentrating our study on the Bcl-2 family of proteins, in particular the Bax protein, which represents a non-reversible trigger for apoptosis. There are many challenges in studying Bax that have to be overcome. This is due to its mitochondria membrane association after apoptosis initiation, multi-component complexes that it can form, and the time dependent changes in its conformation and physiological properties following the apoptosis signal. This requires new methodologies and approaches in Nuclear Magnetic Resonance in order to allow us to study this protein in its various physiological forms at a detailed molecular level and with a high efficiency. We recently determined the structure of Bax and the Bim-BH3 peptide complex. Our structure showed a distinct initiation site of Bax by Bim-BH3, challenging the current model of Bcl-2 protein regulation that has been in place for more than a decade. Based on our model we designed various in vitro mitochondria and in vivo cell based assays to unequivocally confirm our findings. In addition we initiated studies of mitochondria fission. We solved the structures of yeast and human Fis1 protein, a component that recruits other members of the fission machinery. Our structures showed variation in regulatory mechanism of Fis1 in yeast compared to human. These studies provided a starting point to investigate protein complexes that regulate mitochondria fission in both yeast and human. We have developed several new NMR methods to improve efficiency in obtaining structural and relaxation data with increased precision. We evaluated the temperature dependence of backbone 15N and 13C relaxation data to show that backbone correlated motion does not exist. This is important in interpreting NMR measurements involving these nuclei for structural information. We illustrated the presence of non-trivial NMR spin relaxation interferences that could affect the outcome of scalar coupling measurements. We then showed that one could take advantage of 13C relaxation data to refine NMR structure. In addition we developed a new way to analyze dipolar coupling data and proposed a way to acquire different relaxation times simultaneously in a single experiment. In order to evaluate the validity of our NMR relaxation analysis, we developed stochastic simulation of slow domain motion. We have successfully determined residue specific 15N and 13C chemical shift anisotropy (CSA) of a protein in solution using molecular alignment. This allowed us to correlate various components of the CSA tensors to specific structural elements. In the process we had to create an alternative means of protein alignment by the use of type I collagen matrix. We developed a new method in using solvent and protein interaction information as structural restraints. We showed that this approach enhanced the convergence of our NMR structure calculation. Furthermore, we also illustrated that this information can be used to validate NMR structures. This technology is very useful in determining structures of protein complexes. We are finalizing our structure of the complex between Bax and a peptide from a retrovirus viral protein vMIA. Our structure is unique and helps to explain the sequestration of Bax from activation in the apoptosis pathway. It also indirectly suggests the possible structural regions of Bax which is important for membrane insertion and its polymerization. At the same time, in collaboration with Youle's lab we showed that cys mutants could be designed to preserve the structure of Bax in the cytosol of the cell. We used NMR to show that the conformation of the mutant protein is the same as the wild type in solution. This mutant protein actually helped in showing that Bax is actually in equilibrium between cytosol and mitochondria membrane. By changing this equilibrium thru protein-protein interaction, apoptosis can be initiated. In addition, we have initiated a study of another Bcl-2 protein Bid which is cleaved prior to apoptosis induction. We are studying the truncated form of Bid (t-Bid) in a membrane environment to determine its structure and hope that it will shed lights on how it interacts with other Bcl-2 proteins: Bax and Bcl-xL on the mitochondria membrane during apoptosis.
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