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UBC Theses and Dissertations

Synthesis and characterization of template-assembled three- and four- helix bundle proteins Causton, Ashley Scott

Abstract

The unique three-dimensional structure of a protein is the result of a multitude of non-covalent interactions within the polypeptide backbone, and between the side chains of its constituent amino acids. One method that can be used to simplify and study the noncovalent forces present in protein structure is to use a template assembled synthetic protein (TASP): This method employs peptide strands that are attached to a template which directs them to fold into a pre-determined folding pattern. [Diagram of Three-Helix Bundle TASP] [Diagram of Four-Helix Bundle TASP] This thesis studies three- and four-helix bundles using the TASP concept. The templates are cyclotribenzylene ("CTB") and cavitand bowl ("bowl") macrocycles, which have three- and four-fold symmetry, respectively. Amphiphilic peptides (for example, CEELLKKLEELLKKG) which were designed to form helical bundles, were attached to the templates via disulfide bonds. The CTB and bowl TASPs were synthesized by first activating the side chain of a cysteine residue in the peptide strand, and then reacting it with a benzyl thiol moiety on the template. TASPs containing various peptide sequences were investigated in terms of their structure and stability. The CTB and bowl TASPs were found to have helical structure, and enhanced stability to chemical denaturation, exhibiting the co-operative unfolding that is a characteristic of natural proteins. The initial CTB and bowl TASPs contained peptides with the template-bound cysteine residue adjacent to the "helix" sequence, and were found to undergo self-association. This was attributed to incorrect bundling of the constituent helices, resulting from a template-to-helix linker that was too short. Therefore, flexible glycine residues were introduced between the template-bound cysteine residue and the helix in the attached polypeptides. A three-glycine "spacer" (i.e. CGGGEELLKKLEELLKKG) was found to be optimal for reducing TASP self-association. The ultimate goal of this research is to produce TASPs with "native-like" conformational specificity. This first generation of CTB and bowl TASPs appeared to have molten globule structure, which is consistent with other de novo designed proteins that contained a degenerate series of amino acids in their core; this is a good starting point for future designs. By varying the size and shape of the central residue in the TASP's helices, some difference in their stability was observed. These studies demonstrated the validity of these TASP models for investigating protein structure.

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