| Stability and specificity of transmembrane domain self-association by In this thesis, I investigate the sequence dependence of homodimerization of the transmembrane domain of the pro-apoptotic C. elegans protein BNIP3. Using site directed mutagenesis and two assays for dimerization, I show that the tight association of the CeBNIP3 transmembrane domain relies on overlapping but distinct sets of residues depending on the assay: in membranes, the critical residues are N183xxSFxxxGxxxG 194, whereas in detergents, the key residues are S186FxxGxxxGxxxS 198. The small residue Ser 186, the bulky residue Phe 187, and small residues Gly 190 and Gly 194 play key roles in CeBNIP3 dimerization in both assays. However, CeBNIP3 TMD self-association in detergents, but not membranes, depends critically on Ser 198; self-association in lipid bilayers, but not detergents, depends on Asn 183. Comparison with the previously identified dimerization motif for the human BNIP3 ortholog (SHxxAlxxGlxxG) shows that the residues that drive CeBNIP3 dimerization in membranes are chemically similar to, but distinct from, those that drive HsBNIP3 association.;To explore how interfacial BNIP3 residues determine dimer stability and specificity, I generated a combinatorial library from the CeBNIP3 and HsBNIP3 motifs, (STT)(H/N)xx(A/S)(I/F)xxG(I/A)xxG, and tested the hybrid sequences for dimerization. All combinations of interfacial residues support strong to extremely strong dimerization in membranes, suggesting that the two parental sequences adopt similar structures. Not all sequences form dimers in detergents, and dimerization propensity correlates weakly with sequence hydrophobicity. Manipulating the solvent conditions to enhance the hydrophobic effect increases dimerization of some sequences but not others.;The CeBNIP3 and HsBNIP3 transmembrane domains form homodimers but not heterodimers in detergents. Hybrid motif sequences show differing propensities to form heterodimers with wildtype CeBNIP3 TMD and HsBNIP3 TMD: some hybrids discriminate, binding only one wildtype sequence, and some interact with both. My findings identify the sequence elements responsible for stability and specificity of BNIP3-type transmembrane domain dimerization. My results also show that the hydrophobicity of membrane spans strongly influences their behavior in detergent assays of protein-protein interactions. The demonstration that altering the aqueous solvent conditions can improve the stability of integral membrane proteins in detergents may be of general importance in membrane protein biochemistry. |
