Sevil Weinkauf

Icosahedral macromolecules have a wide spectrum of potential nanotechno­logical applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150 Å icosahedral capsid consisting of 60 subunits and crystallizes in space group P6₃22 or C2. However, the quality of these crystals is poor and structural information is only available at 2.4 Å resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit-cell parameters a = b = 313.02, c = 365.77 Å, = = 90.0, = 120°, and diffracts to 1.6 Å resolution.

SecA is the peripheral membrane-associated subunit of the enzyme complex 'preprotein translocase' which assists the selective transport of presecretory proteins into and across bacterial membranes. The SecA protein acts as the molecular motor that drives the translocation of presecretory proteins through the membrane in a stepwise fashion concomitant with large conformational changes accompanying its own membrane insertion/retraction reaction cycle coupled to ATPase activity. The high flexibility of SecA causes a dynamic conformational heterogeneity which presents a barrier to growth of crystals of high diffraction quality. As shown by fluorescence spectroscopy, the T(m) of the endothermic transition of cytosolic SecA from Bacillus subtilis is shifted to higher temperatures in the presence of 30% glycerol, indicating stabilization of the protein in its compact membrane-retracted conformational state. High glycerol concentrations are also necessary to obtain three-dimensional crystals suitable for X-ray diffraction analysis, suggesting that stabilization of the conformational dynamics of SecA may be required for 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 A at 100 K and diffract X-rays to approximately 2.70 A using a high-flux synchrotron-radiation source.