Porin activity and sequence analysis of a 31-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp1).
We have recently reported the isolation and purification of the Treponema pallidum outer membrane and the identification of its rare protein constituents, including a 31-kDa protein markedly enriched in the outer membrane preparation (D.R. Blanco, K. Reimann, J. Skare, C.I. Champion, D. Foley, M. M. Exner, R. E. W. Hancock, J. N. Miller, and M. A. Lovett, J. Bacteriol. 176:6088-6099, 1994). In this study, we report the cloning, sequencing, and expression of the structural gene which encodes the 31-kDa outer membrane protein, designated Tromp1. The deduced amino acid sequence from the tromp1 gene sequence encodes a 318-amino-acid polypeptide with a putative 40-amino-acid signal peptide. Processing of Tromp1 results in a mature protein with a predicted molecular mass of 30,415 Da and a calculated pI of 6.6. Secondary-structure predictions identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of Tromp1 containing 14 transmembrane segments is proposed. Specific antiserum against a recombinant Tromp1 fusion protein was generated and was used to identify native Tromp1 in cellular fractionation. Upon Triton X-114 extraction and phase separation of T. pallidum, the 31-kDa Tromp1 protein was detected in the detergent-phase fraction but not in the protoplasmic cylinder or aqueousphase fractions, consistent with a hydrophobic outer membrane protein. Anti-Tromp1 antiserum was also used to identify native Tromp1 purified from whole T. pallidum by Triton X-100 solubilization followed by nondenaturing isoelectric focusing. Reconstitution of purified Tromp1 into planar lipid bilayers showed porin activity based on the measured single channel conductanes of 0.15 and 0.7 nS in 1 M KCl. These findings demonstrate that Tromp1 is a transmembrane outer membrane porin protein of T. pallidum.
Blanco
D R
DR
Department of Microbiology and Immunology, School of Medicine, University of California at Los Angeles 90024, USA.
Champion
C I
CI
Exner
M M
MM
Erdjument-Bromage
H
H
Hancock
R E
RE
Tempst
P
P
Miller
J N
JN
Lovett
M A
MA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D003902:Detergents; D005798:Genes, Bacterial; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D017830:Octoxynol; D011092:Polyethylene Glycols; D018272:Porins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D014210:Treponema pallidum / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D003902:Detergents; D008051:Lipid Bilayers; D018272:Porins; D011994:Recombinant Proteins; C093836:Tromp1 protein, Treponema pallidum; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40