H-1 AND N-15 NMR-STUDY OF HUMAN LYSOZYME

The N-15 signal assignment of human lysozyme was carried out by using H-1-H-1 and H-1-N-15 two dimensional experiments. To solve the severe overlap problem of the NH signals, uniform labeling of the protein with N-15 was introduced. The uniformly N-15 labeled protein was prepared using a high-expression system of Saccharomyces cerevisiae. From the analyses of H-1 and N-15 NMR spectra, all of the backbone N-15 signals of the molecule were assigned to each specific residue in the amino acid sequence. Recently published proton signal assignments [Redfield & Dobson (1990) Biochemistry, 29, 7201-7214] were confirmed by these complementary data. In addition, assignments were extended to side chain (NH2)-N-15 groups of asparagine and glutamine. Elements of secondary structure were deduced from the pattern of sequential and medium-range NOE connectivities. Two beta-sheets and four alpha-helices could be identified in the protein, which were in good agreement with those determined by X-ray crystallography. The interaction between human lysozyme and its inhibitor N-acetyl-chitotriose was investigated by N-15-H-1 HMQC spectra. Most of the N-15-NH cross-peaks in the spectra were separated well enough to be followed during the titration experiment. Residues whose NH proton signals decrease in intensity upon complex formation, are located mainly around subsites B, C, and D. Local conformational changes were observed around the fourth helix adjacent to the cleft of human lysozyme.