Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae.
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.
Parge
H E
HE
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Bernstein
S L
SL
Deal
C D
CD
McRee
D E
DE
Christensen
D
D
Capozza
M A
MA
Kays
B W
BW
Fieser
T M
TM
Draper
D
D
So
M
M
Getzoff
E D
ED
Tainer
J A
JA
D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification* / Q000648:ultrastructure; D003460:Crystallization; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000648:ultrastructure*; D007525:Isoelectric Focusing; D009344:Neisseria gonorrhoeae / Q000648:ultrastructure*; D011487:Protein Conformation; D014961:X-Ray Diffraction
D001425:Bacterial Outer Membrane Proteins; D036781:Fimbriae Proteins