Unfolding Pathways of Individual Bacteriorhodopsins

Abstract

Atomic force microscopy and single-molecule force spectroscopy were combined to image and manipulate purple membrane patches from Halobacterium salinarum. Individual bacteriorhodopsin molecules were first localized and then extracted from the membrane; the remaining vacancies were imaged again. Anchoring forces between 100 and 200 piconewtons for the different helices were found. Upon extraction, the helices were found to unfold. The force spectra revealed the individuality of the unfolding pathways. Helices G and F as well as helices E and D always unfolded pairwise, whereas helices B and C occasionally unfolded one after the other. Experiments with cleaved loops revealed the origin of the individuality: stabilization of helix B by neighboring helices.

Forced Unfolding Mechanism of Bacteriorhodopsin as Revealed by Coarse-Grained Molecular Dynamics

“…They proposed, however, a velocity-dependent unfolding mechanism based on the kinetics of an intramolecular interaction network. Oesterhelt et al (4) selectively cleaved the E-F loop and extracted the truncated bR chain from the C-terminal end of helix E. The truncated chain exhibited similar F-D curves to those of the wild-type except for the lack of the single peak that corresponds to helices F-G. Likewise, Kedrov et al (8) performed a similar experiment of sodium proton antiporter A, another transmembrane a-helical protein.…”

Direct mapping of the solid–liquid adhesion energy with subnanometre resolution

“…Atomic force microscopy's 7 (AFM) ability of visualizing the topography and the property of surfaces and interfaces at a molecular level 8 has enabled a rapid development in the understanding of surface phenomena. Its versatility allows the exploration of hard and soft materials in vacuum 9-12 , in air 13 , but also in complex liquids 14,15 , often allowing imaging at sub-nanometre and sometimes atomic resolution 16 . In dynamic mode 17 (vibrating cantilever), AFM has proven sensitive to the interfacial compliance of viscous liquids and provided quantitative information about the structure of liquid layers between the AFM tip and the solid surface 18 , with, in some cases, atomic resolution 15 .…”

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

“…Membrane proteins are a perfect example of this case. Mechanical pulling of single bacteriorhodopsins from their native membrane induces the step-wise unfolding of the protein structure (Oesterhelt et al 2000). In contrast to titin, where the unfolding event of individual domains is probability-driven, the secondary structure elements of a membrane protein unfold sequentially with each unfolding event resulting in characteristic unfolding peak.…”

“…Probing mechanical stability of NhaA Many of above insights on molecular interactions being established within membrane proteins have been originally revealed on bacteriorhodopsin (Müller et al 2002c;Oesterhelt et al 2000). At the same time, most of these insights could also be obtained on other membrane proteins, such as halorhodopsin from H. salinarum (Cisneros et al 2005), human aquaporin-1 from red blood cells (Moller et al 2003), bovine rhodopsin from native disc membranes (Sapra et al, submitted), or NhaA from E. coli .…”

“…Then, we discuss recent advances in SMFS performed on membrane proteins. Such experiments enabled detecting the stability of various membrane proteins (Oesterhelt et al 2000;Moller et al 2003;Kedrov et al 2004;Cisneros et al 2005), probing their energy landscape ) and refolding kinetics (Kedrov et al 2005b). Protein structures were observed to contain stable segments such as those represented by transmembrane α-helices, polypeptide loops or fragments thereof.…”