Structural analysis of an outer surface protein from the Lyme disease spirochete, Borrelia burgdorferi, using circular dichroism and fluorescence spectroscopy.
The etiological agent of Lyme disease is the tick-borne spirochete, Borrelia burgdorferi. A major antigen of B. burgdorferi is a 31 kDa lipoprotein called outer surface protein A (OspA). Recently, a truncated form of OspA (lacking 17 amino acids at the N-terminus) was cloned, expressed and purified in large quantities (Dunn, J.J., Lade, B.A. and Barbour, A.G. (1990) Protein Expression and Purification 1, 159-168). The truncated protein (OspA-257) is water-soluble, retains the ability to bind antibodies from the sera of Lyme disease patients and may prove useful in development of a vaccine against Lyme disease. We have used far UV circular dichroism (CD) and fluorescence spectroscopy to characterize the secondary structure of and to study conformational changes in OspA-257. CD spectra from 260 to 178 nm predict five classes of secondary structure: alpha-helix (11%), anti-parallel beta-sheet (32%), parallel beta-sheet (10%), beta-turns (18%) and aperiodic structures (including 'random coil') (30%). Analysis of the primary sequence of OspA yielded the most likely sites for alpha-helical regions (residues 100-107, 121-134, 253-273) and for antigenic determinants (Lys-46, Asp-82, Lys-231). CD spectra of the native protein show little change from pH 3 to 11. Thermal denaturation curves, indicate that 'salt bridges' play a role in stabilizing the native protein. Both thermal and chemical denaturations that eliminate all secondary structure as judged by CD or fluorescence are reversible. Denaturation by guanidine-HCl (gdn-HCl) appears to be a cooperative, two-state transition, as indicated by a sudden change in the CD spectrum at approximately 0.75 M gdn-HCl, and an isodichroic point at 208 nm in all CD spectra measured from 0.0-1.75 M gdn-HCl.
France
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Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Kieleczawa
J
J
Dunn
J J
JJ
Hind
G
G
Sutherland
J C
JC
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000276:immunology; D000954:Antigens, Surface / Q000737:chemistry* / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D001428:Bacterial Vaccines; D025065:Borrelia burgdorferi; D015748:Borrelia burgdorferi Group / Q000737:chemistry* / Q000276:immunology; D002942:Circular Dichroism; D008074:Lipoproteins; D008193:Lyme Disease / Q000276:immunology; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D008074:Lipoproteins; D008565:Membrane Proteins; C072045:OspA protein