Abstract
Proteins are dynamic and constantly subjected to random perturbations coming from surrounding molecules such as solvent/water and lipid environments. Activities of proteins are often affected by the dynamical structures of the surrounding molecules, but the interactions at a molecular level remain largely unresolved. This dissertation focuses on the characterization of the molecular interactions between protein and its surrounding molecules using spectroscopic tools including electron spin resonance (ESR) and fluorescence methods. In Chapter 1, general background information and a summary of the studies are presented. In Chapter 2, a combined method of advanced ESR techniques and mesoporous materials are presented. We show that because of the improvements in revealing the local structures of hydration molecules by the combined method, molecular details of the interfacial hydration layers near to the biomolecular surface are better revealed, providing new insights into the protein-water interactions. Our result provides evidence for the existence of different hydration layers along a biomolecular surface normal when confined in the nanochannels of the mesoporous materials. The causes and implications concerning the coupled interfacial hydration layers are discussed. In Chapter 3, we report our study on the role of cardiolipin (CL) in regulating the apoptotic activities of B-cell lymphoma-2 associated X (BAX) protein when being associated with membrane lipids. BAX is a proapoptotic member in the BCL2 protein family, which plays an essential role in the mitochondria-mediated apoptosis. BAX is a monomeric cytosolic protein in a healthy cell. Upon apoptotic stress, BAX is induced to go through substantial conformational changes, followed by a translocation to mitochondrial outer membrane, oligomerization, and permeabilization of the membrane. As the oligomerization can occur in either the presence or absence of lipid environments, the role of the lipids in determining the assembly of the various activated forms of BAX (including monomer and dimer unites) has been mysterious. The study presented in Chapter 3 investigated the conformation and assembly of the activated membrane-associated BAX oligomers using several ESR and fluorescence spectroscopic tools. As CL is a signature lipid of mitochondria, much attention is paid to how the apoptotic activity and oligomerization are affected by the composition of CL in lipid environments. We show that, depending on lipid composition, the membrane-associated BAX can exist in a variety of forms, all of which are comprised of BAX dimers whose structures are similar to the reported dimeric BH3-in-groove model. Only in the presence of CL in the membrane can BAX assemble into oligomers to induce a large (> 70 kDa) and stable proteolipidic pore, otherwise BAX forms amorphous aggregates, which only induce relatively small membrane pores. Our result has revealed new insights into the formation of apoptotic BAX oligomers in the mitochondrial membrane and identified the molecular composition of the permeabilizing structure that occurs in the mitochondria-mediated apoptosis.