Morris, C; (2015) Conformationally-assisted chemical reactions using a split protein. Doctoral thesis , UCL (University College London).

Preview

PDF 25-03-15_Caroline_Morris_UCL_thesis.pdf.REDACTED.pdf Download (7MB)

Abstract

The aim of the project was to develop a novel method of protein labelling based upon split proteins. The proposed method would label a target protein via a small peptide sequence expressed at its N-terminus that is selectively targeted by its partner peptide carrying a tag. Split proteins were considered effective for a proximity-assisted labelling method because of the specificity of the peptide fragments for one another, and the extensive background literature available. The project has studied bovine ribonuclease A (RNase A), chymotrypsin inhibitor 2 (CI2) and calbindin D9k, three well-documented split proteins. In each instance the sequence was divided into two peptides that were synthesized by Fmoc- SPPS, with the exception of the S-protein of RNase A which was commercially available. Conformationally-assisted ligation with a peptide N-(Me)sulfonamide was initially explored using literature precedent, and the affinity of the interaction at this site was also measured by bio-layer interferometry (BLI) or UV/VIS spectroscopy. With limited success in attempts with both RNase A and CI2, calbindin D9k was chosen because of the high affinity reported in the literature between its peptides. Their interaction was assessed by BLI and their secondary structure in the presence of either calcium or a chelator was compared by circular dichroism. In this system conformationally-assisted ligation was observed along with proximity-assisted acyl transfer of a small peptide tag, with promising results. The peptides were also observed to enable click chemistry, when synthesized as a peptide with C-terminal azide and a peptide with N-terminal alkyne, with no requirement for a copper additive. The results described are successful applications of conformationally-assisted chemical reactions, enabled by the specificity and high affinity imparted by the split protein peptides. The reactions are chemoselective and active at biologically relevant concentrations, and are encouraging for the future development of a novel protein labelling method.

Download activity - last month

Export as XMLCSVJSON

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item