Formation of the single-layer β-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain

Borrelia outer surface protein A (OspA) contains a unique single-layer P-sheet that connects N and C-terminal globular domains. This singlelayer beta -sheet segment (beta -strands 8-10) is highly stable in solution, although it is exposed to Me solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer P-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to P-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta -strand. H-1-N-15 heteronuclear NOE measurements and amide H-H-2 exchange revealed that the single-layer beta -sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta -sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta -sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta -sheet structure. (C) 2001 Academic Press.