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Volume 272, Number 16, Issue of April 18, 1997 pp. 10710-10720
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Structural and Functional Characterization of a Recombinant PorB Class 2 Protein from Neisseria meningitidis
CONFORMATIONAL STABILITY AND PORIN ACTIVITY

(Received for publication, January 3, 1997)

Conceição A. S. A. Minetti , Joseph Y. Tai , M. S. Blake , Jeffrey K. Pullen , Shu-Mei Liang and David P. Remeta ^¶

From  North American Vaccine, Inc., Beltsville, Maryland 20705 and ^¶ NHLBI, National Institutes of Health, Bethesda, Maryland 20892

An outer membrane PorB class 2 protein from Neisseria meningitidis has been overexpressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of zwitterionic detergent. The purified recombinant and native (strain M986) counterpart exhibit most of the typical functional and structural properties that are characteristic of bacterial porins. Channel forming activity has been monitored by incorporating class 2 into reconstituted liposomes and measuring the permeation rates of various oligosaccharides through the proteoliposomes to derive a pore diameter of ~1.6 nm. Structural studies employing a combination of spectroscopic and electrophoretic techniques reveal that recombinant and native class 2 are identical in terms of overall conformational stability. Both proteins form stable trimers in zwitterionic detergent and retain significant secondary and tertiary structure in the presence of SDS. The thermal unfolding of zwittergen-solubilized class 2 trimers (T_m = 88 °C) is reversible and characterized by solvent exposure of aromatic residues with concomitant disruption of tertiary and partial loss of secondary structures. SDS-induced destabilization and irreversible unfolding of the native trimeric assembly occurs at temperatures above 60 °C. Our physicochemical studies of PorB class 2 protein furnish significant insight regarding the structural and functional properties of this meningococcal outer membrane protein within the porin superfamily.

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