title: Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor.
abstract: Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and Rfree of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β2αβ fold characteristic for Kazal-family serine proteinase inhibitors.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Edyta
authors.initials: E
authors.lastname: Kopera
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Bal
authors.affiliation: Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia.
authors.forename: Martina
authors.initials: M
authors.lastname: Lenarčič Živkovič
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Angela
authors.initials: A
authors.lastname: Dvornyk
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Kludkiewicz
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Krystyna
authors.initials: K
authors.lastname: Grzelak
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; NanoBioMedical Centre, A. Mickiewicz University, Poznan, Poland.
authors.forename: Igor
authors.initials: I
authors.lastname: Zhukov
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Włodzimierz
authors.initials: W
authors.lastname: Zagórski-Ostoja
authors.affiliation: Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland; Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Mariusz
authors.initials: M
authors.lastname: Jaskolski
authors.affiliation: Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland.
authors.forename: Szymon
authors.initials: S
authors.lastname: Krzywda
mesh_terms: D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D018360:Crystallography, X-Ray / Q000379:methods; D019286:Endopeptidase K / Q000037:antagonists & inhibitors; D019476:Insect Proteins / Q000032:analysis / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000648:ultrastructure*; D016415:Sequence Alignment; D020860:Subtilisin / Q000037:antagonists & inhibitors
chemical_list: D000345:Affinity Labels; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; C425550:silk proteinase inhibitor 2, Galleria mellonella; D020860:Subtilisin; D019286:Endopeptidase K