Francesca Muzio, Antonio J. Astarita, Meredith Buono and Josiah Morris

Abstract:

A study of the conformational changes of proteins is important in understanding biological activity. “Open” and “closed” forms of proteins can regulate normal protein activity while mutations in the protein amino acid sequence can cause conformational changes that negatively affect normal function. We aim to use fluorine-19 NMR (FNMR) spectroscopy to investigate protein conformational changes. This technique involves incorporating fluorinated aromatic amino acids into proteins of interest using bacterial expression techniques. Even though fluorine nuclei are not natively found in proteins, they can be incorporated without disturbing the structure or activity of the target protein. As a proof-of-principle, we have successfully expressed glutathione S-transferase (GST) in minimal media with fluorine-labeled indole derivatives, which are biosynthesized into the protein as fluorinated tryptophan residues. We have also developed a methodology to characterize the fluorine-labeled proteins using mass spectrometry and FNMR and plan to apply this technique to the study of other proteins of interest.