TY - JOUR
T1 - Structural analysis of an outer surface protein from the Lyme disease spirochete, Borrelia burgdorferi, using circular dichroism and fluorescence spectroscopy
AU - France, Louisa Lee
AU - Kieleczawa, Jan
AU - Dunn, John J.
AU - Hínd, Geoffrey
AU - Sutherland, John C.
N1 - Funding Information:
We thank Ann Emrick, Denise Monteleone and John Trunk for assistance with the experiments and analysis of data, and Dr. Erwin London (State University of New York at Stony Brook) for the use of his Spex 212 fluorolog fluorometer. We also thank Dr. Curtis W. Johnson and his colleagues at Oregon State University for providing a copy of VARSELEC, the program for the analysis of protein secondary structure. Research supported by the Office of Health and Environmental Research and the Division of Energy Biosciences, Office of Basic Energy Sciences (JK and GH), United States Department of Energy (USDOE) and a grant from the National Institutes of Health (GM34662) to JCS. The National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory is supported by the Office of Chemical Research and The Office of Materials Research, USDOE. The circular dichroism spectrometer and fluorometer at port U9B of the NSLS are supported by the Office of Health and Environmental Research, USDOE.
PY - 1992/3/27
Y1 - 1992/3/27
N2 - 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: α-helix (11%), anti-parallel β-sheet (32%), parallel β-sheet (10%), β-turns (18%) and aperiodic structures (including 'random coil') (30%). Analysis of the primary sequence of OspA yielded the most likely sites for α-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 ∼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.
AB - 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: α-helix (11%), anti-parallel β-sheet (32%), parallel β-sheet (10%), β-turns (18%) and aperiodic structures (including 'random coil') (30%). Analysis of the primary sequence of OspA yielded the most likely sites for α-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 ∼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.
KW - Circular dichrosim
KW - Fluorescence spectroscopy
KW - Lyme disease
KW - Outer surface protein
KW - Protein structure
KW - Spirochete
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U2 - 10.1016/0167-4838(92)90424-C
DO - 10.1016/0167-4838(92)90424-C
M3 - Article
C2 - 1554741
AN - SCOPUS:0026593789
SN - 0167-4838
VL - 1120
SP - 59
EP - 68
JO - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
JF - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
IS - 1
ER -