Protein <sup>19</sup>F NMR in <i>Escherichia coli</i>
2010-01-13T00:00:00Z (GMT) by
Although overexpression and <sup>15</sup>N enrichment facilitate the observation of resonances from disordered proteins in <i>Escherichia coli</i>, <sup>15</sup>N 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 <sup>19</sup>F-labeled proteins. Resonances from small (∼10 kDa) globular proteins containing the amino acid analogue 3-fluoro-tyrosine can be observed in cells, but for larger proteins the <sup>19</sup>F 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 <sup>19</sup>F NMR.
CC BY-NC 4.0