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J. Biol. Chem., Vol. 265, Issue 4, 2278-2285, 02, 1990
HE Parge, SL Bernstein, CD Deal, DE McRee, D Christensen, MA Capozza, BW Kays, TM Fieser, D Draper and M So
Pilus fibers are long protein filaments on many pathogenic bacteria that
participate in attachment to host cells. Although the self- assembling
protein pilin is the major structural component of the Neisseria
gonorrhoeae pilus fiber, several other proteins co-purified with pilin
through the repeated solubilization-reassociation steps of the biochemical
purification. Pilin solubilized in the nondenaturing detergent
n-octyl-beta-D-glucopyranoside remained an aggregate of about 100 kDa at pH
9.5, but was reduced to a 40-kDa dimer at pH 10.5, suggesting that assembly
involves electrostatic interactions of lysine, tyrosine, or other side
chains with high pKa values. Pilin dimers and aggregates of higher
molecular mass were partially stable even in the presence of sodium dodecyl
sulfate and beta-mercaptoethanol. Removal of pilus-associated proteins and
stabilization of pilin multimers permitted the reproducible crystallization
of pilin. Three-dimensional needle- and plate-shaped crystals of purified
N. gonorrhoeae pilin (strain MS11 variant C30) grew from 36 to 40%
polyethylene glycol 400, pH 8.0-9.0, in space group C222, with cell
dimensions a = 126.4, b = 121.2, c = 26.7 A and Vm = 2.84 A3/dalton for one
molecule per asymmetric unit. The best crystals diffracted to 2.4 A
resolution using synchrotron radiation, were stable to x-ray damage, and
appear suitable for determination of the atomic structure. This approach of
stabilizing and crystallizing an intermediate assembly state may be useful
for other fiber-forming proteins, which have previously not been
successfully crystallized in forms that diffract to atomic resolution.
Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
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