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Default Incorporation of trifluoromethionine into a phage lysozyme: implications and a new ma

Incorporation of trifluoromethionine into a phage lysozyme: implications and a new marker for use in protein 19F NMR.

Related Articles Incorporation of trifluoromethionine into a phage lysozyme: implications and a new marker for use in protein 19F NMR.

Biochemistry. 1997 Mar 18;36(11):3404-16

Authors: Duewel H, Daub E, Robinson V, Honek JF

Much interest is currently focused on understanding the detailed contribution that particular amino acid residues make in protein structure and function. Although the use of site-directed mutagenesis has greatly contributed to this goal, the approach is limited to the standard repertoire of twenty amino acids. Fluorinated amino acids have been utilized successfully to probe protein structure and dynamics as well as point to the importance of specific residues to biological function. In our continuing investigations on the importance of the amino acid methionine in biological systems, the successful incorporation of L-S-(trifluoromethyl)homocysteine (L-trifluoromethionine; L-TFM) into bacteriophage lambda lysozyme (LaL), an enzyme containing three methionine residues, is reported. The L isomer of TFM was synthesized in an overall yield of 33% from N-acetyl-D,L-homocysteine thiolactone and trifluoromethyl iodide. An expression plasmid giving strong overproduction of LaL was prepared and transformed into an Escherichia coli strain auxotrophic 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 initially grown in a Met-rich media followed by protein induction under careful 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 high (70%) and low (31%) levels of TFM incorporation. TFM-LaL at both levels of incorporation exhibited analogous activity to the wild type enzyme and were inhibited by chitooligosaccharides indicating that incorporation of the analogue did not hinder enzyme function. Interestingly, the 19F solution NMR spectra of the TFM-labeled enzymes consisted of four sharp resonances spanning a chemical shift range of 0.9 ppm, with three of the resonances showing very modest shielding changes on binding of chitopentaose. The 19F NMR analysis of TFM-LaL at both high and low levels of incorporation suggested that one of the methionine positions gives rise to two separate resonances. The intensities of these two resonances were influenced by the extent of incorporation which was interpreted as an indication that subtle conformational changes in protein structure are induced by incorporated TFM. The similarities and differences between Met and TFM were analyzed using ab initio molecular orbital calculations. The methodology presented offers promise as a new approach to the study of protein-ligand interactions as well as for future investigations into the functional importance of methionine in proteins.

PMID: 9116020 [PubMed - indexed for MEDLINE]



Source: PubMed
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