The Oms66 (p66) protein is a Borrelia burgdorferi porin.
In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.
Skare
J T
JT
Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, College Station 77843, USA. jskare@tamu.edu
Mirzabekov
T A
TA
Shang
E S
ES
Blanco
D R
DR
Erdjument-Bromage
H
H
Bunikis
J
J
Bergström
S
S
Tempst
P
P
Kagan
B L
BL
Miller
J N
JN
Lovett
M A
MA
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000502:physiology; D001426:Bacterial Proteins; D015748:Borrelia burgdorferi Group / Q000737:chemistry* / Q000502:physiology; D004553:Electric Conductivity; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D018272:Porins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008081:Liposomes; D008565:Membrane Proteins; C107893:Oms66 protein, Borrelia; D018272:Porins