title: 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.
abstract: 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 protein thermostability and folding kinetics. An extra mass of 178 Da arising from the post-translational modification at the N-terminal His tag was observed. The spontaneous alpha-N-6 gluconoylation at the amino group of the His-tagged SH3 domain contributed to the observed extra mass. The partial modification 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 both wild-type and mutant src SH3. Both the wild type and the W43I mutant were crystallized by hanging-drop vapor diffusion and are in the hexagonal space group P6(5)22 with one molecule in the asymmetric unit. Data sets were collected to 1.8 and 1.95 A resolution for the the wild type and the W43I mutant, respectively.
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Kim
authors.forename: E C
authors.initials: EC
authors.lastname: Yi
authors.forename: D
authors.initials: D
authors.lastname: Baker
authors.forename: K Y
authors.initials: KY
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003460:Crystallization; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D019061:src-Family Kinases