STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF A RECOMBINANT PORB CLASS-2 PROTEIN FROM NEISSERIA-MENINGITIDIS - CONFORMATIONAL STABILITY ANDPORIN ACTIVITY

An outer membrane PorB class 2 protein from Neisseria meningitidis has been overexpresed 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 o f bacterial porins. Channel forming activity has been monitored by inc orporating class 2 into reconstituted liposomes and measuring the perm eation rates of various oligosaccharides through the proteoliposomes t o derive a pore diameter of similar to 1.6 nm, Structural studies empl oying a combination of spectroscopic and electrophoretic techniques re veal that recombinant and native class 2 are identical in terms of ove rall conformational stability, Both proteins form stable trimers in zw itterionic detergent and retain significant secondary and tertiary str ucture in the presence of SDS. The thermal unfolding of zwittergen-sol ubilized class 2 trimers (T-m = 88 degrees C) is reversible and charac terized by solvent exposure of aromatic residues with concomitant disr uption of tertiary and partial loss of secondary structures. SDS-induc ed destabilization and irreversible unfolding of the native trimeric a ssembly occurs at temperatures above 60 degrees 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.