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J. Biol. Chem., Vol. 279, Issue 51, 53747-53754, December 17, 2004

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Topology of the Outer Membrane Usher PapC Determined by Site-directed Fluorescence Labeling^*

Nadine S. Henderson, Stephane Shu Kin So, Cheryl Martin, Ritwij Kulkarni, and David G. Thanassi

From the Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York 11794-5120

In contrast to typical membrane proteins that span the lipid bilayer via transmembrane -helices, bacterial outer membrane proteins adopt a -barrel architecture composed of antiparallel transmembrane -strands. The topology of outer membrane proteins is difficult to predict accurately using computer algorithms, and topology mapping protocols commonly used for -helical membrane proteins do not work for -barrel proteins. We present here the topology of the PapC usher, an outer membrane protein required for assembly and secretion of P pili by the chaperone/usher pathway in uropathogenic Escherichia coli. An initial attempt to map PapC topology by insertion of protease cleavage sites was largely unsuccessful due to lack of cleavage at most sites and the requirement to disrupt the outer membrane to identify periplasmic sites. We therefore adapted a site-directed fluorescence labeling technique to permit topology mapping of outer membrane proteins using small molecule probes in intact bacteria. Using this method, we demonstrated that PapC has the potential to encode up to 32 transmembrane -strands. Based on experimental evidence, we propose that the usher consists of an N-terminal -barrel domain comprised of 26 -strands and that a distinct C-terminal domain is not inserted into the membrane but is located instead within the lumen of the N-terminal -barrel similar to the plug domains encoded by the outer membrane iron-siderophore uptake proteins.

Received for publication, August 11, 2004 , and in revised form, September 27, 2004.

^* This work was supported by Public Health Service Grant GM62987 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Tables I–III.

Present address: Dept. of Medicine, Stony Brook University, Stony Brook, NY 11794-8151.

To whom correspondence should be addressed: 242 Center for Infectious Diseases, Stony Brook University, Stony Brook, NY 11794-5120. Tel.: 631-632-4549; Fax: 631-632-4294; E-mail: David.Thanassi{at}stonybrook.edu.

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