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Title: Evaluation of sequential chemoselective peptide ligation and molecular dynamics simulations as tools for the total synthesis of proteins : an example using bovine pancreatic ribonuclease A
Author: Council, Claire Elizabeth
ISNI:       0000 0004 2749 9569
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2013
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Chemoselective ligation between two peptides can be used to produce synthetic peptides and proteins that cannot easily be synthesised as single peptides by solid phase peptide synthesis. Chemoselective reaction between an aldehyde and hydrazine or hydroxylamine, masking the aldehyde as a 1, 2-amino alcohol, has been investigated as a method of sequential ligation that will allow synthesis of individual peptides in high yield and purity, enabling more than two peptides to be ligated. Protected amino acids were used as precursors for the synthesis of 1, 2-amino alcohol derivatives that could be used in solid phase peptide synthesis for the production of peptides bearing a C-terminal 1, 2-amino alcohol, as a masked aldehyde for use in ligation reactions. Limitations of this method led to alternative investigations, using peptides bearing an N-terminal serine as a masked aldehyde, and a C-terminal hydrazide. Bovine pancreatic ribonuclease A was chosen as an example protein for sequential ligation and using this method, peptides were synthesised in high yield and purity for use in ligation reactions. Trial ligation reactions with short test peptides were performed successfully, however problems were experienced during ligations using peptide fragments of ribunuclease due to involvement of the cysteine side chains. Methods to overcome these unwanted reactions resulted in insoluble peptide fragments. Computer modelling using molecular dynamics simulations has been used to investigate the effect of replacing native peptide bonds in ribonuclease on the structure of the protein. The method of molecular dynamics simulation was validated through comparison of the structure of a mutant of ribonuclease from experimental NMR data to the structure produced after a molecular dynamics simulation. Results of the modelling simulations suggest that replacement of native peptide bonds with the chemoselective bond formed through reaction of an aldehyde and hydrazine will have only minor implications for the structure of ribonuclease, and therefore should only have a small impact on enzyme activity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available