High-speed atomic force microscopy (HS-AFM) is becoming a reference tool for the study of dynamic biological processes. The spatial and time resolutions of HS-AFM are on the order of nanometers and milliseconds, respectively, and allow structural and functional characterization of biological processes at the single-molecule level. In this work we present contact-mode HS-AFM movies of purple mem...

We theoretically and experimentally analyze the formation of thick Purple Membrane (PM) polyacrylamide (PA) films by means of optical spectroscopy by considering the absorption of bacteriorhodopsin and scattering. We have applied semiclassical quantum mechanical techniques for the calculation of absorption spectra by taking into account the Fano effects on the ground state of bacteriorhodopsin....

The kinetics and stoi~hiometry of lint-indu~d proton release in purple membrane sus~nsions have been investigated using the pH-indicator dye pyranine and sin~5tu~over flash spectroscopy at a time resolution of 0.1 ps. The number of protons detected by pyranine is inversely proportional to the buffehng power of the medium and 1.1 f 0.2 protons are released per photocycling bacteriorhodopsin mole...

The role of the electron spin in chemistry and biology has received much attention recently owing to to the possible electromagnetic field effects on living organisms and the prospect of using molecules in the emerging field of spintronics. Recently the chiral-induced spin selectivity effect was observed by electron transmission through organic molecules. In the present study, we demonstrated t...

Archaerhodopsin-2 (aR2), the sole protein found in the claret membrane of Halorubrum sp. Aus-2, functions as a light-driven proton pump. In this study, structural analysis of aR2 was performed using a novel three-dimensional crystal prepared by the successive fusion of claret membranes. The crystal is made up of stacked membranes, in each of which aR2 trimers are arranged on a hexagonal lattice...

The effect of deuteration on the 13C linewidths of U-13C, 15N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in 2H medium (for both 50:50% 1H:2H, and ...

Bacteriorhodopsin (bR) trimers naturally form two-dimensional hexagonal crystals in purple membrane (PM), which make it very stable. However, the dnaturation of bR was found to occur during a very narrow pH range when the pH was increased above 12.0, as indicated by inactivation of the photochemical cycle observed by flash photolysis kinetic spectra. Here, atomic force microscopy was used to st...

In atomic force microscopy (AFM), finding sparsely distributed regions of interest can be difficult and time-consuming. Typically, the tip is scanned until the desired object is located. This process can mechanically or chemically degrade the tip, as well as damage fragile biological samples. Protein assemblies can be detected using the back-scattered light from a focused laser beam. We previou...

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 extractio...

Multiple molecular dynamics simulations of bacterioopsin pulling from its C-terminus show that its alpha-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 Asp-212 and Lys-216 on helix G an...