Studies of exchangeable hydrogens in lysozyme by means of Fourier transform proton magnetic resonance

Abstract

Fourier transform proton magnetic resonance techniques have been used to observe the solvent exchangeable hydrogens of hen and human lysozymes. Spectra of samples in both D$_{2}$O and H$_{2}$O were obtained, the spectra in H$_{2}$O being recorded while selectively saturating the H$_{2}$O resonance with an applied radio frequency field. Difference spectroscopy has shown that there are no detectable differences in conformation between the proteins in D$_{2}$O and H$_{2}$O. Difference spectroscopy has also been used to follow the exchange of hydrogens with solvent (D$_{2}$O) and to observe the resonances of the exchangeable (NH) hydrogens separately from those of the non-exchangeable (CH) ones. The resonances from the indole C2 and N1 protons of a tryptophan, residue 108, have been firmly assigned in the spectra of both hen and human lysozyme by using lanthanide ion shift and relaxation probes in conjunction with crystallographic information. These assignments have been confirmed by studying the effects of pH on the resonances. This study has revealed an interesting interaction between the residues tryptophan 108 and glutamic acid 35. The structures of the active sites of the two lysozymes examined in solution are very similar to each other and to their crystal structures.