A fundamental step in understanding protein structure and function is determining the precise roles of key, individual amino acids in the polypeptide chain. This includes the roles of amino acids involved in direct contact with other molecules, including substrates and ligands, and those that are modified, for example by phosphorylation, as a means for modulating protein function. In this project, both prokaryotic and eukaryotic systems for the precise insertion of either phosphoserine or pyrrolysine at desired sites within a polypeptide chain will be developed. The insertion of phosphoserine can be used to probe the effects of specific phosphorylation events on protein structure and function. The insertion of pyrrolysine can be used to probe the role of individual lysine residues in ligand binding and in catalysis. These systems rely on the development of unique archael tRNA synthetase/tRNA suppressor pairs capable of inserting phosphoserine or pyrrolysine at engineered, nonsense codons both in vitro and in vivo. The broader impacts of this project is significant because success in this venture would be transforming, providing new tools for researchers in biophysics, biochemistry, cell biology, metabolic biochemistry, and other fields throughout the life sciences. The participation of a graduate student researcher and a high school science teacher is planned in aspects of this work, as well.
