INCORPORATION OF TRIFLUOROMETHIONINE INTO A PHAGE LYSOZYME - IMPLICATIONS AND A NEW MARKER FOR USE IN PROTEIN F-19 NMR

Much interest is currently focused on understanding the detailed contr ibution that particular amino acid residues make in protein structure and function. Although the use of site-directed mutagenesis has greatl y contributed to this goal, the approach is limited to the standard re pertoire of twenty amino acids. Fluorinated amino acids have been util ized successfully to probe protein structure and dynamics as well as p oint to the importance of specific residues to biological function. In our continuing investigations on the importance of the amino acid met hionine in biological systems, the successful incorporation of L-S-(tr ifluoromethyl)homocysteine (L-trifluoromethionine; L-TFM) into bacteri ophage lambda lysozyme (LaL), an enzyme containing three methionine re sidues, is reported. The L isomer of TFM was synthesized in an overall yield of 33% from N-acetyl-D,L-homocysteine thiolactone and trifluoro methyl iodide. An expression plasmid giving strong overproduction of L aL was prepared and transformed into Escherichia coli strain auxotroph ic for methionine permitting the expression of LaL in the presence of L-TFM. The analogue would not support growth of the auxotroph and was found to be inhibitory to cell growth. However, cells that were initia lly grown in a Met-rich media followed by protein induction under care ful control of the respective concentrations of L-Met and L-TFM in the media, were able to overexpress TFM-labeled LaL (TFM-LaL) at both hig h (70%) and low (31%) levels of TFM incorporation. TFM-LaL at both lev els of incorporation exhibited analogous activity to the wild type enz yme and were inhibited by chitooligosaccharides indicating that incorp oration of the analogue did not hinder enzyme function. Interestingly, the F-19 solution NMR spectra of the TFM-labeled enzymes consisted of four sharp resonances spanning a chemical shift ran of 0.9 ppm, with three of the resonances showing very modest shielding changes on bindi ng of chitopentaose. The F-19 NMR analysis of TFM-LaL at both high and low levels of incorporation suggested that one of the methionine posi tions gives rise to two separate resonances. The intensities of these two resonances were influenced by the extent of incorporation which wa s interpreted as an indication that subtle conformational changes in p rotein structure are induced by incorporated TFM. The similarities and differences between Met and TFM were analyzed using ab initio molecul ar orbital calculations. The methodology presented offers promise as a new approach to the study of protein-ligand interactions as well as f or future investigations into the functional importance of methionine in proteins.