Effect of a mutation at arginine 301 on the stability, crystal quality andthe preliminary crystallographic analysis of recombinant canavalin from Canavalia ensiformis

The technique of site-directed mutagenesis was used to implement rational a mino acid changes in the plant storage protein canavalin, the major seed st orage protein of the jack bean (Canavali ensiformis). The mutations were ta rgeted to amino acids previously demonstrated to be involved in either the intra- or intermolecular salt bridges, thought to be responsible for mainta ining the three-dimensional structure of the trimer. The amino acid changes were designed to disrupt the salt bridge interactions by substituting a ne utral alanine for a negatively charged aspartate or glutamate, or by substi tuting a negatively charged glutamate for a positively charged arginine. Th e resulting recombinant mutants were subsequently expressed, purified, and crystallized. The crystals of the mutant versions of canavalin were compare d to those of the wild-type canavalin by visual inspection and X-ray analys is. Of the crystals obtained for the mutants, those for the Arg301Glu mutat ion appeared to be more stable with fewer surface defects than any of the o ther mutants or the wild-type protein. The I/sigma ratio of reflections ver sus the resolution for the Arg301Glu mutation was approximately 30% greater over the entire resolution range than that obtained for any of the other m utations or for the wild-type. Additionally, the crystals of Arg301Glu muta tions displayed lower mosaicity. Finally, the Arg301Glu mutation displayed a striking increase in the transition temperature when subjected to thermal denaturation. This paper describes the rationale and techniques behind the mutation of canavalin and suggests possible explanations for the observed and measured differences between the Arg301Glu mutant and the wild-type pro tein. We show the initial crystallographic structure analysis of this mutan t and its preliminary implications. (C) 2001 Elsevier Science B.V. All righ ts reserved.