Proteins made by improved chemical methods unveil the molecular basis of protein structure

An original set of chemical methods has been developed and was applied to the total synthesis of crambin to show their utilities. The three peptide segment ligation was optimized, and the total synthesis of crambin by the three segments ligation gave a ten-fold increase in yield (Chapter 2). A 'one-pot' method was developed to eliminate the tedious purification steps and consquent handling losses (Chapter 3). Three peptides could be ligated in one-pot, and folded in the same reaction mixture to give desired protein molecule with only a single final purification step. The His 6-tag assisted chemical protein synthesis was developed. The presence of the His6 tag enabled the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification (Chapter 4). A kinetically controlled convergent synthesis was devised by applying a very simple and general concept, a kinetic control of reactivity, to the convergent synthesis of proteins (Chapter 5).; Chemically synthesized proteins were used to improve our understanding of the functional role of a salt bridge in the crambin molecule, and our understanding of the chemical basis of the amino acid residues found in the C' position of C-cap of an alpha-helix. The role of a salt bridge in the crambin molecule was investigated, and I showed that the salt bridge of crambin molecule guides the formation of correct disulfide bonds (Chapter 6). The total synthesis of a protein analogue of novel topology, 'crambin cyclotide' was explored, and I observed that cysteine residues found their unique disulfide bond partners in the presence of an artificial covalent bond (Chapter 7). The enantiomeric protein D-crambin was crystallized with exactly opposite handedness compared to the L-crambin (Chapter 8). A striking conservation of crystal structure between a ubiquitin molecule containing D-amino acid and a corresponding wild type ubiquitin molecule was observed (Chapter 9). By the use of ubiquitin diastereomers, I showed that the preference for the C' Gly termination at the C-cap of protein alpha-helix is based primarily on conformation effects and to a much less extent on solvation effects (Chapter 10).