Studies on an Artificial Trypsin Inhibitor Peptide Derived from the Mung Bean Trypsin Inhibitor: Chemical Synthesis, Refolding, and Crystallographic Analysis of Its Complex with Trypsin1

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Abstract
The active fragment with Lys at the reactive site of mung bean trypsin inhibitor (MBILF) is composed of two peptide chains, Al of 26 residues and A2 of 9 residues linked via two disulfide bonds. In the present study, a peptide of 22 residue comprising the sequence of chain Al from position 3 to 24 was synthesized by the solid-phase method. This synthetic peptide with six Cys residues contains a reactive site at position LysllI-Serl2I (I denotes an inhibitor residue). Air oxidation and HPLC purification resulted in two antitrypsin active components, SPC1 and SPC2. Neither SPC1 nor SPC2 can stoichiometrically inhibit trypsin. The K1 values of SPC1 and SPC2 are 1.2×l0–7 and 4.0×10–8 M, respectively. The complexes of SPC1 and SPC2 with bovine β-trypsin (BTRY) were crystallized by ammonium sulphate precipitation at pH 6.4 and 6.0, respectively. The two crystals have the same crystal form with space group P212121 and cell dimension of a=63.22 A, b = 63.56 A, and c = 69.84 A. The crystal structure of one complex, SPC1-BTRY, was determined and refined at 2.2 A resolution to a final R-value of 19.2%. From the resulting electron density map, 9 residues of SPC1, from position 91 to 171, were identified clearly and three-dimension atomic model of the 9-residue reactive loop formed by a disulfide bridge, Cys91-Cys17I, was built. No electron density corresponding to the other 13 residues was observed in the present map. The refined atomic coordinates of this complex and structure factors has been deposited with the Brookhaven Protein Data Bank (reference: SMF).

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