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 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.
Koide
S
S
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA. Shohei_Koide@urmc.rochester.edu
Bu
Z
Z
Risal
D
D
Pham
T N
TN
Nakagawa
T
T
Tamura
A
A
Engelman
D M
DM
D000954:Antigens, Surface / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001428:Bacterial Vaccines; D015748:Borrelia burgdorferi Group / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D008074:Lipoproteins; D009682:Magnetic Resonance Spectroscopy; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D008074:Lipoproteins; C072045:OspA protein