New rapid method of protein crystal structure analyzes making use of protein engineering techniques

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The aim was to develop a new method useful for both the solution of the phase problem and interpretation of the resultant electron density map making use of protein engineering techniques. Specifically, in this study, the protein engineering techniques were used to prepare potein specimens containing selenomethionyl residues in place of natural methionyl residues.Making use of these techniques, we solved three-dimensional structures of two proteins, the BphC enzyme (250K dalton ; J.Mol. Biol. 255,735-752 (1996)) and the BphD enzyme (250K dalton ; manuscript in preparation) both working in a metabolic pathway for biphenyl and its derivatives including the notorious environmental pollutant, PCB.The following observation as to the present method have been made.1) In so far as the X-ray diffraction intensity measurement is done using conventional laboratory (rather than synchrotron) X-rays (without anomalous dipersion data), the phase determination solely based on the selen sites (the so-called SIR method) is not strong eough to lead to a final protein structure solution.2) Still, the phase information derived as above can improve the quality of the rough phase information provided by other heavy-atom derivatives. Furthermore, for improved phase information obtained in this way, various modern techniques of electron density improvement (such as the solvent-flattening method) can be as effective as to lead to a fianl protein structure solution.