1H nuclear magnetic resonance studies of hen lysozyme-N-acetylglucosamine oligosaccharide complexes in solution. Application of chemical shifts for the comparison of conformational changes in solution and in the crystal.
Two-dimensional 1H nuclear magnetic resonance spectroscopy has been used to examine the complexes formed in solution between hen egg-white lysozyme and N-acetylglucosamine (GlcNAc) oligosaccharides. Changes in chemical shift have been measured for resonances of the majority of residues of lysozyme on binding the monomer, dimer and trimer of GlcNAc. The three inhibitors induce very similar changes in chemical shift, and these increase slightly with the length of the oligosaccharide. The largest changes are confined principally to the vicinity of site C in the active site cleft of the enzyme. These changes in chemical shift have been compared with differences in the ring current chemical shifts calculated from the crystal structures of unbound and GlcNAc3 bound lysozyme. This comparison suggests that the major conformational changes of residues in the vicinity of site C of the enzyme, that are caused by the binding of GlcNAc3, observed in the diffraction studies are at least consistent with the changes that occur in solution. Small changes in chemical shift are observed in the enzyme in regions remote from the active site, which indicate that the effects of inhibitor binding are felt throughout the enzyme. These changes in chemical shift correlate to a lesser extent than those near site C with the changes in chemical shift predicted from changes in conformation observed in the crystal structures. The results illustrate that chemical shifts are useful in assessing the significance of small conformational changes in proteins, although the usefulness of this approach will be limited by the resolution of the crystallographic structures, as well as the uncertainties in the origins of the chemical shift. Although conformational changes in site C account for many of the changes in the NMR spectrum of lysozyme, evidence is, however, presented for multiple binding sites for the GlcNAc oligosaccharides in solution, perhaps involving partial occupancy of site D.
Lumb
K J
KJ
Oxford Centre for Molecular Sciences, University of Oxford, U.K.
Cheetham
J C
JC
Dobson
C M
CM
D000117:Acetylglucosamine / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry*; D011485:Protein Binding; D012996:Solutions; D013696:Temperature
D009844:Oligosaccharides; D012996:Solutions; D009113:Muramidase; D000117:Acetylglucosamine