Post-translational modification of the N-terminal His tag interferes with the crystallization of the wild-type and mutant SH3 domains from chicken src tyrosine kinase

Structural studies of the wild type and mutants of the src SH3 domain were initiated to elucidate the correlation of the native-state topology with pr otein thermostability and folding kinetics. An extra mass of 178 Da arising from the post-translational modification at the N-terminal His tag was obs erved. The spontaneous alpha -N-6 gluconoylation at the amino group of the His-tagged SH3 domain contributed to the observed extra mass. The partial m odification of the N-terminal His-tag produced heterogeneity, both in size and in charge, in the Escherichia coli expressed SH3 domain. The removal of the His tag from the SH3 domain was essential for the crystallization of b oth wild-type and mutant src SH3. Both the wild type and the W43I mutant we re crystallized by hanging-drop vapor diffusion and are in the hexagonal sp ace group P6(5)22 with one molecule in the asymmetric unit. Data sets were collected to 1.8 and 1.95 Angstrom resolution for the the wild type and the W43I mutant, respectively.