Conformational stabilization and crystallization of the SecA translocation ATPase from Bacillus subtilis.
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.
Weinkauf
S
S
Institut für Technische Chemie, Technische Universität München, Lichtenbergstrasse 4, D-85748 Garching, Germany. sevil.weinkauf@ch.tum.de
Hunt
J F
JF
Scheuring
J
J
Henry
L
L
Fak
J
J
Oliver
D B
DB
Deisenhofer
J
J
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000302:isolation & purification; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D005990:Glycerol / Q000378:metabolism; D026901:Membrane Transport Proteins; D011487:Protein Conformation; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D005990:Glycerol