Unfolding mechanics of multiple OspA substructures investigated with single molecule force spectroscopy

J Mol Biol. 2003 Nov 7;333(5):993-1002. doi: 10.1016/j.jmb.2003.09.010.


We investigated mechanical unfolding of Borrelia burgdorferi outer surface protein A (OspA), a Lyme disease antigen containing a unique single-layer beta-sheet, with atomic force microscopy (AFM). We mechanically stretched a monomeric unit, rather than a tandem repeat, by pulling it from its N and C-terminal residues without using intervening polymer as a spacer. We detected two peaks in the force-extension profile before the final rupture of a fully extended polypeptide, which we interpreted as unfolding of multiple substructures in OspA. The double-peaked unfolding curves are consistent with results of previous thermodynamic studies showing two cooperative units in OspA. The mechanical unfolding processes were reversible, and the two substructures refolded within one second. Mutations near the boundary of the two thermodynamic cooperative units reduced the height of the first unfolding peak to undetectable levels and marginally affected the second one, indicating that the boundary between the two mechanical substructures is related to that previously assigned between the thermodynamic cooperative units. Based on a "worm-like chain" analysis of our AFM data, we propose a model for mechanical unfolding of OspA, where nearly a half of the chain is stretched with minimal resistive force, followed by sequential breakdown of C-terminal and N-terminal substructures. Based on these results, we discuss similarities and differences between mechanical and thermodynamic unfolding reactions of OspA. This work demonstrates that AFM study of monomeric proteins can elucidate details of the intramolecular mechanics of protein substructures.

Publication types

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

MeSH terms

  • Animals
  • Antigens, Surface / chemistry
  • Antigens, Surface / metabolism*
  • Bacterial Outer Membrane Proteins / chemistry
  • Bacterial Outer Membrane Proteins / metabolism*
  • Bacterial Vaccines
  • Borrelia burgdorferi / chemistry
  • Borrelia burgdorferi / metabolism
  • Humans
  • Lipoproteins*
  • Microscopy, Atomic Force
  • Mutation
  • Protein Denaturation
  • Protein Structure, Tertiary


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