Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 376, Issue 4, Pages 1076-1090Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2007.12.027
Keywords
atomic force microscopy; energy landscape; Hammond effect; single-molecule force spectroscopy; transition states
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Funding
- NIGMS NIH HHS [R01 GM063919, R01 GM063919-07, R01 GM063919-08] Funding Source: Medline
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Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a Simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wildtype and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others. (C) 2007 Elsevier Ltd. All rights reserved.
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