Successful recovery of the normal electrophysiological properties of PorB (Class 3) porin from Neisseria meningitidis after expression in Escherichia coli and renaturation

• Published in: Biochimica et biophysica acta 1998-03, Vol.1370 (2), p.289-298
• Main Authors: Song, Jiming, Minetti, Conceição A.S.A., Blake, M.S., Colombini, Marco
• Format: Article
• Language: English
• Subjects: Bacterial Outer Membrane Proteins - biosynthesis; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - physiology; Electrophysiological property; Electrophysiology; Escherichia coli - genetics; Ion Channel Gating; Ion Channels - metabolism; Ion Channels - physiology; Membrane Lipids - metabolism; Membrane Lipids - physiology; Membrane Potentials; Membranes, Artificial; Neisseria; Neisseria meningitidis - genetics; Neisseria meningitidis - growth & development; Outer membrane; Porin; Porins; Recombinant; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Voltage-gating
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/S0005-2736(97)00279-4

Neisseria meningitidis PorB class 3 porins obtained either from native membranes (wild-type) or recovered from inclusion bodies following expression in Escherichia coli (recombinant), have been reconstituted into solvent-free planar phospholipid membranes. The wild-type and recombinant porins exhibited the same single-trimer conductance (1–1.3 nS in 200 mM NaCl), tri-level closure pattern, characteristic of functional channel trimers, and pattern of insertion into planar membranes. Both proteins were open at low voltages and displayed two voltage-dependent closure processes, one at positive and the other at negative potentials. Both showed asymmetric voltage dependence such that one gating process occurred at lower voltages ( V o=15 mV) than the other ( V o=25 mV). The sign of the potential that resulted in closure at low voltages varied from membrane to membrane indicating that they may have the property of auto-directed insertion (in analogy to the mitochondrial channel, VDAC). In the case of the recombinant porin, the steepness of the voltage dependence of one gating process was slightly less ( n=1.3) than that observed for the other process or for the wild-type channel ( n=1.5–1.7). Both channels have a high (40%) probability of closure even at 0 mV. While both channels show a slight selectivity for Cl − over Na +, the selectivity of the recombinant porin is a bit higher (permeability ratio of 2.8 vs. 1.6) as measured using a 2-fold salt gradient. Thus, the method employed to refold the recombinant porin was successful in not only restoring wild-type structure [H.L. Qi, J.Y. Tai, M.S. Blake, Expression of large amounts of Neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers, Infect. Immun. 62 (1994) 2432–2439; C.A.S.A. Minetti, J.Y. Tai, M.S. Blake, J.K. Pullen, S.M. Liang, D.P. Remeta, Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity, J. Biol. Chem. 272 (1997) 10710–10720] but also the overall electrophysiological function.