Conformational stabilization and crystallization of the SecA translocationATPase from Bacillus subtilis

SecA is the peripheral membrane-associated subunit of the enzyme complex 'p reprotein translocase' which assists the selective transport of presecretor y proteins into and across bacterial membranes. The SecA protein acts as th e molecular motor that drives the translocation of presecretory proteins th rough the membrane in a stepwise fashion concomitant with large conformatio nal changes accompanying its own membrane insertion/retraction reaction cyc le coupled to ATPase activity. The high flexibility of SecA causes a dynami c conformational heterogeneity which presents a barrier to growth of crysta ls of high diffraction quality. As shown by fluorescence spectroscopy, the Tm of the endothermic transition of cytosolic SecA from Bacillus subtilis i s shifted to higher temperatures in the presence of 30% glycerol, indicatin g stabilization of the protein in its compact membrane-retracted conformati onal state. High glycerol concentrations are also necessary to obtain three -dimensional crystals suitable for X-ray diffraction analysis, suggesting t hat stabilization of the conformational dynamics of SecA may be required fo r effective crystallization. The SecA crystals grow as hexagonal bipyramids in the trigonal space group P3(1)12; they possess unit-cell parameters a = 130.8, b = 130.8, c = 150.4 Angstrom at 100 K and diffract X-rays to appro ximately 2.70 Angstrom using a high-flux synchrotron-radiation source.