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

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 alpha-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 alpha-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.