Complex stability of single proteins explored by forced unfolding experiments

¹ BioTechnological Center, University of Technology Dresden

^* To whom correspondence should be addressed. E-mail: harald.janovjak{at}biotec.tu-dresden.de.

Submitted on January 20, 2005
Revised on February 15, 2005
Accepted on 21 March 2005

	Abstract

In the last decade atomic force microscopy (AFM) 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 (Fn), 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 (MC) 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.

Key Words: AFM, folding, force spectroscopy, membrane protein, single molecule, stability

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