Protein 19F NMR in Escherichia coli

Although overexpression and N-15 enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, N-15 enrichment alone is insufficient for detecting most globular proteins. Here, we explain this dichotomy and overcome the problem while extending the capability of in-cell NMR by using F-19-labeled proteins. Resonances from small (similar to 10 kDa) globular proteins containing the amino acid analogue 3-fluoro-tyrosine can be observed in cells, but for larger proteins the F-19 resonances are broadened beyond detection. Incorporating the amino acid analogue trifluoromethyl-L-phenylalanine allows larger proteins (up to 100 kDa) to be observed in cells. We also show that site-specific structural and dynamic information about both globular and disordered proteins can be obtained inside cells by using F-19 NMR.