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dc.contributor.authorPacker, Laura Een_US
dc.date.accessioned2015-08-05T01:18:14Z
dc.date.available2015-08-05T01:18:14Z
dc.date.issued2012
dc.date.submitted2012
dc.identifier.other(ALMA)contemp
dc.identifier.urihttps://hdl.handle.net/2144/12578
dc.descriptionThesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.en_US
dc.description.abstractAnalysis of mutations within the hydrophobic core of excised domains of proteins yields insight towards the determinants of protein structure, specificity, and stability. Two systems with hydrophobic cores were tested: (1) villin headpiece and (2) the N-terminal helical region of apolipoprotein B (apoB). In the headpiece system, our hypothesis was that mutation of the residues in the unusual buried salt bridge to strictly hydrophobic residues would increase its stability. The villin headpiece domain (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved buried salt bridge between residues E39 and K70 connects the N- and C-terminal subdomains. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations are made in a stabilized mutant of HP67, H41Y. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin headpiece. The second tested hypothesis was that subtle mutations of hydrophobic residues within the N-terminal α-helical domain of apolipoprotein B (apoB) would significantly affect its tertiary structure. ApoB is the protein component on low density lipoprotein (LDL) particles. High levels of LDL (the "bad" cholesterol) are associated with cardiovascular disease, the leading cause of death in Western countries. There is little direct structural information on any part of apoB and structural studies of the N-terminal domain of apoB are complicated by its aggregation in the absence of lipids. The N-terminal domain of apoB is homologous to lipovitellin, whose structure is known. A construct that encodes the first six helices of the apoB N-terminal α-helical domain (H6) is stable and was chosen to test our hypothesis. Two conservative point mutations (L324M and L343V) within this region of apoB have been shown to cause Familial Hypobetalipoproteinemia (FHBL). FHBL is characterized by low levels of plasma apoB-containing lipoproteins. Chemical unfolding monitored by CD indicates a reduction in stability for the two point mutants. 1H-15N HSQC spectra indicate significant structural changes, suggesting repacking of the helices. We believe the possible change in helical packing of these single point mutants in apoB leads to ER associated degradation (ERAD) and prevents the secretion of VLDL into the blood stream, resulting in FHBL.en_US
dc.language.isoen_US
dc.publisherBoston Universityen_US
dc.titleDeterminants of protein structure, specificity, and stability in excised domains of villin and apolipoprotein Ben_US
dc.typeThesis/Dissertationen_US
etd.degree.nameDoctor of Philosophyen_US
etd.degree.leveldoctoralen_US
etd.degree.disciplineBiophysicsen_US
etd.degree.grantorBoston Universityen_US


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