Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides.
A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 A resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 A. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (VM = 2.5 A3 Da(-1)). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 A resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.
Navarro
Marcos Vicente de A S
MV
Institute of Physics of São Carlos, University of São Paulo, Avenida Trabalhador Sãocarlense 400, CEP 13560-970, São Carlos, SP, Brazil.
Vierira
Débora F
DF
Nagem
Ronaldo A P
RA
de Araújo
Ana Paula U
AP
Oliva
Maria Luiza V
ML
Garratt
Richard C
RC
D036481:Bauhinia / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism; D007454:Iodides / Q000737:chemistry; D007610:Kallikreins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D015233:Models, Statistical; D010455:Peptides / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012974:Sodium Iodide / Q000737:chemistry; D013696:Temperature; D014961:X-Ray Diffraction; D014965:X-Rays
D007454:Iodides; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D007610:Kallikreins; D012974:Sodium Iodide