Multistep denaturation of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet

Biochemistry. 1999 Apr 13;38(15):4757-67. doi: 10.1021/bi982443+.


Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi, is a dumbbell-shaped protein in which two globular domains are connected by a three-stranded beta-sheet segment that is solvent-exposed on both faces. Previous studies showed that the whole protein, including the single-layer beta-sheet, is highly rigid. To elucidate the folding mechanism and the role of the central beta-sheet in the formation of the rigid molecule, we investigated the equilibrium thermal denaturation reaction of OspA. We applied differential scanning calorimetry, heteronuclear NMR spectroscopy, and solution small-angle X-ray scattering (SAXS) to characterize the reaction in detail. All three techniques revealed that OspA denatures in two separable cooperative transitions. NMR measurements on OspA specifically 15N-labeled at Lys residues identified the locations of the two folding units and revealed that the C-terminal segment is less stable than the remaining N-terminal segment. The boundary between the two folding units is located within the central beta-sheet. The interconversion among the three folding states (fully folded, C-terminus unfolded, and fully denatured) is slow relative to chemical shift differences (<24 Hz), indicating that there are significant kinetic barriers in the denaturation reactions. SAXS measurements determined the radius of gyration of the native protein to be 25.0 +/- 0.3 A, which increases to 34.4 +/- 1.0 A in the first transition, and then to 56.1 +/- 1.6 A in the second transition. Thus, the intermediate state, in which the C-terminal folding unit is already denatured, is still compact. These results provide a basis for elucidating the folding mechanism of OspA.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Surface / chemistry*
  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Vaccines
  • Borrelia burgdorferi Group / chemistry*
  • Calorimetry, Differential Scanning
  • Lipoproteins*
  • Magnetic Resonance Spectroscopy
  • Protein Denaturation
  • Protein Folding
  • Scattering, Radiation
  • Spectrometry, Fluorescence


  • Antigens, Surface
  • Bacterial Outer Membrane Proteins
  • Bacterial Vaccines
  • Lipoproteins
  • OspA protein