title: Complex stability of single proteins explored by forced unfolding experiments.
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.
authors.affiliation: BioTechnological Center, University of Technology, 01307 Dresden, Germany. harald.janovjak@biotec.tu-dresden.de
authors.forename: Harald
authors.initials: H
authors.lastname: Janovjak
authors.forename: K Tanuj
authors.initials: KT
authors.lastname: Sapra
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Müller
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry; D001703:Biophysics / Q000379:methods*; D002138:Calibration; D005353:Fibronectins / Q000737:chemistry; D046911:Macromolecular Substances / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods*; D008958:Models, Molecular; D009010:Monte Carlo Method; D016011:Normal Distribution; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D005353:Fibronectins; D046911:Macromolecular Substances; D011506:Proteins; D014867:Water; D001436:Bacteriorhodopsins