English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

Stability of bacteriorhodopsin alpha-helices and loops analyzed by single-molecule force spectroscopy

MPS-Authors
/persons/resource/persons219470

Müller,  D. J.
Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society;

/persons/resource/persons219461

Möller,  C.
Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society;

Locator
There are no locators available
Fulltext (public)
There are no public fulltexts available
Supplementary Material (public)
There is no public supplementary material available
Citation

Müller, D. J., Kessler, M., Oesterhelt, F., Möller, C., Oesterhelt, D., & Gaub, H. (2002). Stability of bacteriorhodopsin alpha-helices and loops analyzed by single-molecule force spectroscopy. Biophysical Journal, 83(6), 3578-3588.


Cite as: http://hdl.handle.net/21.11116/0000-0001-130E-B
Abstract
The combination of high-resolution atomic force microscopy imaging and single-molecule force spectroscopy allows the identification, selection, and mechanical investigation of individual proteins. In a recent paper we had used this technique to unfold and extract single bacteriorhodopsins (BRs) from native purple membrane patches. We show that subsets of the unfolding spectra can be classified and grouped to reveal detailed insight into the individualism of the unfolding pathways. We have further developed this technique and analysis to report here on the influence of pH effects and local mutations on the stability of individual structural elements of BR against mechanical unfolding. We found that, although the seven transmembrane a-helices predominantly unfold in pairs, each of the helices may also unfold individually and in some cases even only partially. Additionally, intermittent states in the unfolding process were found, which are associated with the stretching of the extracellular loops connecting the a-helices. This suggests that polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR. Chemical removal of the Schiff base, the covalent linkage of the photoactive retinal to the helix G, resulted in a predominantly two-step unfolding of this helix. It is concluded that the covalent linkage of the retinal to helix G stabilizes the structure of BR. Trapping mutant D96N in the M state of the proton pumping photocycle did not affect the unfolding barriers of BR.