1H and 15N NMR study of human lysozyme.
The 15N signal assignment of human lysozyme was carried out by using 1H-1H and 1H-15N two dimensional experiments. To solve the severe overlap problem of the NH signals, uniform labeling of the protein with 15N was introduced. The uniformly 15N labeled protein was prepared using a high-expression system of Saccharomyces cerevisiae. From the analyses of 1H and 15N NMR spectra, all of the backbone 15N 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 15NH2 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 15N-1H HMQC spectra. Most of the 15N-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.
Ohkubo
T
T
Protein Engineering Research Institute, Osaka.
Taniyama
Y
Y
Kikuchi
M
M
D000117:Acetylglucosamine; D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009113:Muramidase / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry; D011487:Protein Conformation
D006859:Hydrogen; D009113:Muramidase; D009584:Nitrogen; D000117:Acetylglucosamine