Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme

We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme. By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein. The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances. These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique [Bax, A., Griffey, R. H., & Hawkins, B.L. (1983) J. Magn. Reson. 55, 301-335] which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined. With these approaches, all five phenylalanine amide protons give resolved resonances. Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less. These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments. This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed.