Abstract
Protein folding is one of the most important unsolved questions of structural biology because of the desire to understand the link between the primary sequence and structure. Proteins can fold on a microsecond to millisecond time scale. Fluorescence and IR spectroscopy are very important tools to follow these fast kinetic events, however, natural fluorescent groups in proteins, Tyr and Trp, are not perfect substitutions for most amino acids and new fluorescent probes are needed. In IR spectroscopy the signal from the protein backbone is used to follow protein dynamics. In an IR spectrum, broad and overlapping peaks are generally observed and site-specific IR probes would represent a significant advance. With the advances in molecular biology, it is now possible to introduce new spectroscopic probes into proteins recombinantly.In this dissertation the N-terminal domain of ribosomal protein L9 (NTL9) was used as a model system to investigate the unnatural amino acids, p-cyanophenylalanine (FCN) as a fluorescent and azidohomoalanine (Aha) as an IR-active probe for folding studies. Recently, FCN was shown as a fluorescent and IR probe to study protein dynamics. The fluorescence quantum yield of the probe increases dramatically when it is hydrogen bonded. NTL9-F5FCN is generated by both peptide synthesis and recombinantly by an orthogonal tRNA/tRNA synthetase pair. The folding kinetics of NTL9 is studied with stopped-flow fluorescence. In addition to this, the effect of amino acid side chains on FCN fluorescence is investigated with model peptides in order to use this popular probe accurately.The azido stretching vibration is in a transparent region of the protein IR spectrum and is sensitive to solvation. Aha also has a high extinction coefficient. The azido analog of methionine, Aha is incorporated into proteins by solid-phase peptide synthesis and recombinantly in high yield using methionine auxotrophic strains. Aha was incorporated into two sites in NTL9. The mutations did not perturb the overall fold of the protein. The frequency of the azido mode is observed to undergo a significant blue shift in the thermally unfolded state, indicating that the group provides a sensitive probe of protein folding and sidechain burial.
