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 completing our study on the structure of truncated Bid in a membrane environment. The structure of this activated form of Bid in the membrane is consistent with the make up of the helical nature of this protein. Most helices are interacting with the membrane thru their hydrophobic surface, while their hydrophylic faces are exposed to the solvent. No long range interaction could be observed from the conventional NMR NOE experiments. We probed the overall fold of the protein thru the use of paramagnetic relaxation enhancement experiment. Our data showed that the N- an C-termini arre close to within 10-20 angstrom. This is a accomplished by the bend in the helical chain of tBid. Our findings provided another observation in which we can start to model the molecular events that occur during apoptosis initiation involving the Bcl-2 proteins. In parallel we developed a new protocol in order to measure conformational change of the protein Bax in living cell. We devised a scheme in order to incorporate three different fluorophores into the protein without disturbing its structure. We chose a pair of fluorophores that can allow us to measure FRET distance, while the extra fluorophore was used as a control for environmental change. With this set up we have been able to measure distances in Bax while it is in the cytosol and after its translocated into the mitochondria membrane following apoptosis trigger. Based on our measurements we are proposing the conformations of Bax in the cytosol as well as in the mitochondria membrane after following translocation. Our data showed that Bax C-terminal helix is not tucked into the hydrophobic core even in the cytosol. In contrast other helices in the protein adopt conformations similar to the structure of Bax that was determined in solution. The conformation of the C-terminal helix of Bax in the cytosol is correlated to a slow diffusion phase of the protein that was measured using fluorescence correlation spectroscopy in the live cells. We attributed this slow diffusion phase to a population of Bax that interconverting between cytosolic and mitochondria. Upon translocation we could observe significant changes in distances between helices in Bax. Our measurements indicated that Bax adopted an open conformation in the mitochondria. In addition, we carried out inter-molecular distance measurements between Bax molecules. Our allowed us to conclude that Bax formed oligomers through two distinct contact regions. We also tested a BH3 mimic, ABT737, to see if this has any effect on Bax conformation. Our data showed that ABT737 only accelerated the rate of translocation but no changes in measured distances could be observed relative to the values measured without ABT737. This indicated that ABT737 does not directly influence Bax but could change the rate of translocation by interacting with most likely Bcl-xL. We followed the Bax conformational study in live cell using FRET and FCS measurements to look at Bid conformational changes after initiation of apoptosis. We showed that tBid, Bid that has been cleaved by a caspace, translocates to the mitochondria membrane and adopts an extended structure similar to its NMR structure that we determined in a detergent micelle. We also showed if we cross linked parts of Bid, thus preventing it from converting to an extended conformation, it is no longer translocates to the mitochondria membrane and is inactive. We further probe inter-molecular contacts between tBid molecules as well as between tBid and Bax after the initiation of apoptosis. The tBid forms head to tail oligomer and it furthers form contacts involving the BH3 domain of Bax oligomers. Our data therefore suggests a formation of network of protein oligomer that promotes the permeabilization of the mitochondria outer membrane. In order to determine the orientation of a certain domain relative to the membrane bilayer we developed a hybrid FRET method to detect location of a fluorophore associated with a protein relative to the membrane. We chose to use dipricylamine (DPA) as a FRET acceptor that can be modulated by a pulsed electric field to be close to the inner or outer leaflet of the membrane. Using short acquisition time confocal imaging we can readily determine the location of the fluorophore relative to the bilayer. We used this method to determine the orientation of helix-6 in Bax after it is incorporated into a vesicle.
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