Successful recovery of the normal electrophysiological properties of PorB (class 3) porin from Neisseria meningitidis after expression in Escherichia coli and renaturation.
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 (Vo=15 mV) than the other (Vo=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.
Song
J
J
University of Maryland, Department of Zoology, College Park, MD 20742, USA.
Minetti
C A
CA
Blake
M S
MS
Colombini
M
M
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D004594:Electrophysiology; D004926:Escherichia coli / Q000235:genetics; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism / Q000502:physiology; D008563:Membrane Lipids / Q000378:metabolism / Q000502:physiology; D008564:Membrane Potentials; D008567:Membranes, Artificial; D009345:Neisseria meningitidis / Q000235:genetics / Q000254:growth & development; D018272:Porins; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D008563:Membrane Lipids; D008567:Membranes, Artificial; D018272:Porins; D011994:Recombinant Proteins; C057699:porin protein, Neisseria