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Complex Stability of Single Proteins Explored by Forced Unfolding Experiments

BioTechnological Center, University of Technology, 01307 Dresden, Germany

Correspondence: Address reprint requests and inquiries to Harald Janovjak, Tel.: 49-351-463-40331; Fax: 49-351-463-40342; E-mail: harald.janovjak{at}biotec.tu-dresden.de.

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 -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 -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.

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