Investigation into the relationship between sequence, structure and folding in a model lipocalin: Human serum retinol-binding protein
Date of Award
Doctor of Philosophy (Ph.D.)
Biochemistry and Molecular Biology
First Committee Member
Keith Brew, Committee Chair
Investigations into the determinants of stability and rapid folding were pursued from an evolutionary perspective to test the hypothesis that they are conserved. The nature of sequence conservation in two divergent and functionally diverse protein superfamilies, the lipocalins and immunoglobulins, was systematically elucidated. The analysis identified discrete groups of highly conserved residues, termed evolutionarily conserved regions (ECRs) that comprise approximately 10% of the sequence. Analysis of 3-D structures revealed that these regions are clustered together in the native fold anil consist of both conserved long-range and short-range interactions. Examination of the ECRs in the context of the protein folding problem indicate that they have structural properties similar to those expected in folding nuclei. The existence of prokaryotic and eukaryotic members in both superfamilies suggests that members within a superfamily shared a common ancestor prior to the prokaryotic-eukaryotic split and the conserved regions were selected for early on and have been maintained throughout 2 billion versus of divergent evolution.The human serum retinol-binding protein (RBP) was selected as a model lipocalin for experimental studies. A prokaryotic expression system, purification and in vitro refolding protocols were established to obtain native and functional protein. RBP contains one conserved and three nonconserved tryptophans in the context of the superfamily. Mutants with substitutions for the tryptophans were synthesized to examine the effects of sequence conservation on stability and folding.Thermal denaturation studies reveal that the substitutions to the nonconserved tryptophans (W67, W91, W105) had negligible effect on the Tm (3$\sp\circ$C) as compared to the substitution to the conserved tryptophan (W24) which resulted in a 11$\sp\circ$C decrease. Equilibrium unfolding studies of the native state by chemical denaturation showed that both undergo a reversible two-state transition. Mutations to the conserved tryptophan in comparison to the nonconserved tryptophans significantly effected native state stability by 1.8kcal/mol. RBP undergoes both biphasic folding and unfolding kinetics. The former involves an intermediate with greater fluorescence intensity than the native state and appears to be reported on by Trp105. The studies also indicate that the conserved Trp24, which is a major reporter of the first phase of folding, is buried early in the folding process.
Biology, Molecular; Chemistry, Biochemistry
Greene, Lesley Hope, "Investigation into the relationship between sequence, structure and folding in a model lipocalin: Human serum retinol-binding protein" (1998). Dissertations from ProQuest. 3575.