Sequential unfolding of individual helices of bacterioopsin observed in molecular dynamics simulations of extraction from the purple membrane

¹ University of Modena
² Leeds
³ University of Zurich

^* To whom correspondence should be addressed. E-mail: caflisch{at}bioc.unizh.ch.

Submitted on May 9, 2006
Revised on June 13, 2006
Accepted on 14 July 2006

	Abstract

Multiple molecular dynamics simulations of bacterioopsin pulling from its C-terminus show that its -helices unfold individually. In the first metastable state observed in the simulations helix G is unfolded at its C-terminal segment while the rest of helix G (residues 200-216) is folded and opposes resistance because of a salt bridge network consisting of Asp212 and Lys216 on helix G and Arg82 and Asp85 on helix C. Helix G unfolds inside the bundle because the external force is applied to its C-terminal end in a direction perpendicular to the surface of the membrane. On the contrary, helix F has to flip by 180 degrees to exit from the membrane because the applied force and the helical N-C axis point in opposite directions. At the highest peak of the force, which cannot be interpreted in single-molecule force spectroscopy experiments, helix F has a pronounced kink at Pro186. Mutation of Pro186 and/or the charged side chains mentioned above, which are involved in very favorable electrostatic interactions in the low-dielectric region of the membrane, are expected to reduce the highest peak of the force. Helices E and D unfold in a similar way as helices G and F, respectively. Hence, the force-distance profile and sequence of events during forced unfolding of bacterioopsin are influenced by the up-and-down topology of the seven-helix bundle. The sequential extraction of individual helices from the membrane suggests that the spontaneous (un)folding of bacterioopsin proceeds through metastable bundles of less than seven helices. The metastable states observed in the simulations provide atomic level evidence that corroborates the interpretation of very recent force spectroscopy experiments of bacteriorhodopsin refolding.

Key Words: atomic force microscopy, bacteriorhodopsin, intermediate states, membrane protein folding, molecular dynamics, unfolding pathways

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