Evidence for an alpha-helical epitope on outer surface protein A from the Lyme disease spirochete, Borrelia burgdorferi: an application of steady-state and time-resolved fluorescence quenching techniques.
Outer surface protein A (OspA) is a major antigen of Borrelia burgdorferi, the etiological agent of Lyme disease. A recombinant form of OspA (OspA-257) from B. burgdorferi, strain B31, contains 257 amino acids and a single tryptophan residue at position 216 (Trp-216). Mapping studies indicate that Trp-216 is involved in the epitope for the agglutinating monoclonal antibody 105.5. However, the fluorescence emission maximum of the native protein is 330 nm, indicating that Trp-216 is not solvent-exposed. Primary structure analysis suggests an alpha-helical conformation for residues approx. 204-217, which, if located on the protein surface, would allow Trp-216 to be buried, while leaving hydrophilic residues on the opposite side of the helix exposed. This helix would place Lys-212 within approx. 6 A of Trp-216; the presence of such a positively-charged residue can, in principle, be ascertained from fluorescence quenching studies. Stern-Volmer plots confirm that Trp-216 is indeed buried in the native protein, but is readily accessible to the small polar quencher, acrylamide. Furthermore, the dominant component of the fluorescence emission shows only weak dynamic quenching by the positively-charged quencher, Cs+, while the minor component undergoes static quenching by I-, indicating the proximity of a positively-charged residue. These data are consistent with the existence of an alpha-helix from residues 204-217 in the predicted orientation at the protein surface, hence indicating the structure of the antigenic determinant.
France
L L
LL
Biology Department, Brookhaven National Laboratory, Upton, NY.
Kieleczawa
J
J
Dunn
J J
JJ
Luft
B J
BJ
Hind
G
G
Sutherland
J C
JC
D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001428:Bacterial Vaccines; D025065:Borrelia burgdorferi; D015748:Borrelia burgdorferi Group / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry*; D008074:Lipoproteins; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D000939:Epitopes; D008074:Lipoproteins; C072045:OspA protein; D014364:Tryptophan; D008239:Lysine