High-level expression of uniformly 15N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen lysozyme where phosphate ion binds using NMR measurements

The non-enzymatic deamidation of Asn to Asp is known to occur in proteins and peptides and is accelerated by phosphate buffer [Tyler-Cross, R. and Schirch, V. (1991) J. Biol. Chem. 25, 22549-22556]. We attempted to identify the site in lysozyme where a phosphate ion binds by means of H-1-N-15 HSQC measurements of N-15-labeled lysozyme, which was successfully obtained using Pichia pastoris. As a result, we found that the phosphate ion was preferentially bound to Asn-103 in hen lysozyme, The method presented here may be useful for identifying the binding site of a protein with low molecular weight substances. (C) 1999 Federation of European Biochemical Societies.