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

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia b urgdorferi, 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 mechanis m and the role of the central beta-sheet in the formation of the rigid mole cule, we investigated the equilibrium thermal denaturation reaction of OspA . We applied differential scanning calorimetry, heteronuclear NMR spectrosc opy, and solution small-angle X-ray scattering (SAXS) to characterize the r eaction in detail. All three techniques revealed that OspA denatures in two separable cooperative transitions. NMR measurements on OspA specifically N -15- labeled at Lys residues identified the locations of the two folding un its and revealed that the C-terminal segment is less stable than the remain ing N-terminal segment. The boundary between the two folding units is locat ed within the central beta-sheet. The interconversion among the three foldi ng 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 measuremen ts determined the radius of gyration of the native protein to be 25.0 +/- 0 .3 Angstrom, which increases to 34.4 +/- 1.0 Angstrom in the first transiti on, and then to 56.1 +/- 1.6 Angstrom in the second transition. Thus, the i ntermediate state, in which the C-terminal folding unit is already denature d, is still compact. These results provide a basis for elucidating the fold ing mechanism of OspA.