Single molecule structural studies of bridges, linkers, and hinges in multi-domain proteins: Natural variants, pathological mutants, and therapeutic designs

Nishant Bhasin, University of Pennsylvania

Abstract

Within multi-cellular organisms most proteins contain multiple repeating domains with similar folds but differing physiological function. This thesis seeks to address the structure-function relationship as it pertains to mechanical processes for individual molecules at the level of single tertiary-structure elements and therefore a deepened understanding of local functions within such proteins. Single molecule atomic force microscopy as well as solution studies and modeling are used to study force-driven conformational changes, mechanical stability and extensibility of several multi-domain cytoskeletal proteins. Specifically, the proteins studied here are various constructs of triple-helix family of spectrin proteins (α- and β-spectrin, dystrophin) and β-sheet Ig domain proteins (filamin, titin and VCAM). AFM data on the triple-helix family of spectrin proteins not only confirms the fold of the repeating units but also suggests that the linker between each spectrin repeat is a contiguous helix imparting cooperative stability to the adjoining domains. However linking regions in the β-sheet Ig domain proteins were found to be unstructured, allowing for increased flexibility in Ig multi-domain proteins. Presence of disulfide bridges in Ig domain proteins (VCAM, MelCAM etc.) were shown to limit and further regulate unfolding dictated by microenvironment. Additionally pathogenic mutations within the linker regions were examined to determine the structural basis for altered function. Finally using our structural understanding of the functional 'minimal' dystrophin protein we try to propose a therapeutic design of an efficient and comprehensive gene therapy technique for DMD correction.

Subject Area

Chemical engineering|Biophysics|Biomedical research

Recommended Citation

Bhasin, Nishant, "Single molecule structural studies of bridges, linkers, and hinges in multi-domain proteins: Natural variants, pathological mutants, and therapeutic designs" (2006). Dissertations available from ProQuest. AAI3225429.
https://repository.upenn.edu/dissertations/AAI3225429

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