The rare outer membrane protein, OmpL1, of pathogenic Leptospira species is a heat-modifiable porin.
The outer membranes of invasive spirochetes contain unusually small amounts of transmembrane proteins. Pathogenic Leptospira species produce a rare 31-kDa surface protein, OmpL1, which has a deduced amino acid sequence predictive of multiple transmembrane beta-strands. Studies were conducted to characterize the structure and function of this protein. Alkali, high-salt, and urea fractionation of leptospiral membranes demonstrated that OmpL1 is an integral membrane protein. The electrophoretic mobility of monomeric OmpL1 was modifiable by heat and reduction; complete denaturation of OmpL1 required prolonged boiling in sodium dodecyl sulfate (SDS), 8 M urea, and 2-mercaptoethanol. When solubilized in SDS at low temperature, a small proportion of OmpL1 exhibited an apparent molecular mass of approximately 90 kDa, indicating the existence of an SDS-unstable oligomer. OmpL1 dimers and trimers were demonstrated by nearest neighbor chemical cross-linking. In order to generate purified protein for functional studies, the ompL1 gene was ligated into the pMMB66 expression plasmid under control of the tac promoter. Although expression in Escherichia coli was toxic, most of the OmpL1 produced was found in the outer membrane, as determined by subcellular fractionation. Purified recombinant OmpL1 was reconstituted into planar lipid bilayers, demonstrating an average single channel conductance of 1.1 nS, similar to the major porin activity of native leptospiral membranes. These findings indicate that OmpL1 spans the leptospiral outer membrane and functions as a porin.
Shang
E S
ES
Department of Microbiology & Immunology, UCLA School of Medicine 90024, USA.
Exner
M M
MM
Summers
T A
TA
Martinich
C
C
Champion
C I
CI
Hancock
R E
RE
Haake
D A
DA
D001425:Bacterial Outer Membrane Proteins / Q000502:physiology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004553:Electric Conductivity; D005798:Genes, Bacterial; D006358:Hot Temperature; D007919:Leptospira / Q000235:genetics*; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018272:Porins / Q000235:genetics*; D011994:Recombinant Proteins
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D008051:Lipid Bilayers; D018272:Porins; D011994:Recombinant Proteins; C081904:ompL1 protein, Leptospira