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 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. We confirmed that conformational heterogeneity in Bax in solution, that was proposed as key in its activation, does not exist. We used a battery of biophysical techniques including paramagnetic relaxation enhancement (PRE), residual dipolar coupling (RDC), and double electron-electron resonance (DEER) data. All of our data can fit the original NMR structure of Bax that we first determined. Since all of the Bcl-2 family of proteins contain mostly alpha-helical structure, analyzing their NMR resonances can be a challenge because they don't show large dispersion. We recently developed a new method in employing the additional of lanthanide group to a specific site in a protein that can introduce large dispersion in their NMR resonances. We showed an example where we used such an approach to study NMR signals from unstructured proteins which have very little resonance dispersion. This new technology can be widely used in NMR applications. To better understand the physical property of the lanthanide tags that we used in the above NMR method, we carried out a broad range of experiments to characterize them. We showed that their susceptibility tensor, which is important for introducing pseudo contact shifts, can be modulated by pH and temperature. This behavior can be linked to the apical bound water to the methylated-DOTA caged used in coordinating the lanthanide metal. This information is very useful as we design new and improved compounds to coordinate lanthanide in proteins.
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