Incorporation of Unnatural Amino Acids Via Proteomically Informed Selective Pressure Techniques

Abstract

Determining the three-dimensional structure of a protein using x-ray crystallography can be enhanced by complete or partial incorporation of unnatural amino acids into a target protein. Most naturally occurring atoms do not have sufficient mass to diffract electrons, therefore, heavy atom derivatives of target proteins are required. The Incorporation of unnatural metalloid containing amino acids provide a clear solution to the problem. A proteomic analysis of telluromethionyl Escherichia coli was carried out using the methionine auxotroph E. coli DL41(DE3) (pCock) expression system. Both telluromethionine and methionine exposed cultures were analyzed with the objective of identifying differentially expressed proteins. Analysis was carried out using 2D separation with isoelectric focusing serving in the first dimension and SDS polyacrylamide gel electrophoresis in the second. In-gel tryptic digestion followed by peptide mass fingerprinting using nano-ESI/qTOF/MS allowed identification of differentially expressed proteins. A few of the differentially expressed proteins identified as down-regulated in systems exposed to TeMet were UPase, ZnuA, phosphoserine aminotransferase, and fructose-bisphosphate aldolase. Understanding the biological roles of differentially expressed proteins was used to improve methods of TeMet bioincorporation using the selective pressure technique.