Complex stability of single proteins explored by forced unfolding experiments

Biophys J. 2005 May;88(5):L37-9. doi: 10.1529/biophysj.105.059774. Epub 2005 Mar 25.

Abstract

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of alpha-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of alpha-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins.

MeSH terms

  • Bacteriorhodopsins / chemistry
  • Biophysics / methods*
  • Calibration
  • Fibronectins / chemistry
  • Macromolecular Substances / chemistry
  • Microscopy, Atomic Force / methods*
  • Models, Molecular
  • Monte Carlo Method
  • Normal Distribution
  • Protein Conformation
  • Protein Denaturation
  • Protein Folding
  • Protein Structure, Secondary
  • Proteins / chemistry
  • Water / chemistry

Substances

  • Fibronectins
  • Macromolecular Substances
  • Proteins
  • Water
  • Bacteriorhodopsins