Natural proteins can evolve new three-dimensional structures through mutations in their amino acid sequence. For protein families that exhibit such structural diversity, a major challenge is to understand the scope and nature of structure variation and its relationship to sequence evolution. The Cordes laboratory has begun using transitive homology-based methods to identify, target and structurally characterize natural sequences intermediate between pairs of proteins with distant sequence similarity and different structures. As a proof of principle, this dissertation describes structural studies of two proteins in different families as separate case studies, one involving secondary structure evolution and the other involving topological rearrangement. In the first case, crystallography was applied to solve the structure of a sequence intermediate identified through transitive homology analysis of the Cro transcription regulator family. Comparison with another member resulted in finding two proteins with significant sequence similarity yet different secondary structure compositions and folds. In the second case, transitive homology analysis was applied to look at two members of the insect salivary lipocalins, one with the canonical sequential all-antiparallel β-barrel topology, and another with a unique strand-swapped topology. Three sequence intermediate members were found that each have direct sequence similarity to both topologically distinct relatives. Targeting these sequence intermediate members for structural characterization by NMR led to assignment of the canonical lipocalin topology for one intermediate. The results from these two cases indicate that structurally diverse families may contain members with similar sequences but different folds. As such, transitive homology mapping offers a method to identify and target those members for structural characterization.
|Advisor:||Cordes, Matthew H. J.|
|Commitee:||Ghosh, Indraneel, McEvoy, Megan, Montfort, William|
|School:||The University of Arizona|
|School Location:||United States -- Arizona|
|Source:||DAI-B 72/02, Dissertation Abstracts International|
|Subjects:||Biochemistry, Bioinformatics, Biophysics|
|Keywords:||Cro proteins, Lipocalins, Protein evolution, Protein folding|
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