Charged residues in surface-located loops influence voltage gating of porin from Haemophilus influenzae type b.
Porin of Haemophilus influenzae type b (341 amino acids; M(r) 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24 +/- 0.41 vs. 0.85 +/- 0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50-55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89-91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b.
Arbing
M A
MA
Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada, H3A 2B4.
Dahan
D
D
Boismenu
D
D
Mamer
O A
OA
Hanrahan
J W
JW
Coulton
J W
JW
D000595:Amino Acid Sequence; D002463:Cell Membrane Permeability / Q000502:physiology; D004553:Electric Conductivity; D006193:Haemophilus influenzae / Q000737:chemistry*; D015640:Ion Channel Gating / Q000502:physiology*; D008051:Lipid Bilayers; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D018272:Porins / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D019802:Succinic Acid / Q000737:chemistry
D008051:Lipid Bilayers; D018272:Porins; D019802:Succinic Acid; D008239:Lysine