Book Description

A set of techniques has been developed for the routine synthesis and analysis of proteins by total chemical synthesis. Although the synthesis of proteins has been a goal of organic chemistry for nearly a century, straightforward and reproducible synthesis has remained elusive. Recently, the introduction of chemical ligation techniques for the chemoselective reaction of unprotected peptides in solution has greatly enhanced our ability to synthesize proteins of moderate size. In this thesis, an extension to the chemoselective ligation strategy has been developed which yields a native amide bond at the site of ligation. Native chemical ligation has been used to synthesize several proteins from two unprotected peptide fragments; including the chemokine interleukin 8 and barnase, a 110-residue ribonuclease from Bacillus amyloliquefaciens. Routine synthetic access to protein molecules allows unnatural analogues to be synthesized which may give insight into the molecular basis of protein function. In the second part of this thesis, a system has been developed to systematically study the effect of chemical modifications on the structure and function of a protein. Instead of generating a single protein analogue per synthesis, an array of self-encoded protein analogues can be prepared in a single synthetic procedure. This combinatorial approach was demonstrated by the synthesis of nineteen analogues of the cCrk N-terminal SH3 domain that were folded and analyzed for ligand binding as a single mixture. To read out the composition of the binding and non-binding proteins, a chemical cleavage site was incorporated at the site of modification. Cleavage of this bond created a unique set of peptides that can be identified in a single step by MALDI mass spectrometry. The resulting pattern of peaks relates the position of the chemical modification to its effect on protein function. Taken as a whole, the work presented in this dissertation represents a complete system for the investigation of proteins through chemical synthesis. Using these techniques, proteins of interest can be synthesized, analyzed for functional properties, and then systematically modified to give insight into the molecular basis of protein function.