Interpretation of the spatial charge displacements in bacteriorhodopsin in terms of structural changes during the photocycle

Abstract

We have recently introduced a method, made possible by an improved orienting technique using a combination of electric and magnetic fields, that allows the threedimensional detection of the intramolecular charge displacements during the photocycle of bacteriorhodopsin. This method generates electric asymmetry, a prerequisite for the detection of electric signal on the macroscopic sample, in all three spatial dimensions. Purple membrane fragments containing bacteriorhodopsin were oriented so that their permanent electric dipole moment vectors were perpendicular to the membrane plane and pointed in the same direction. The resulting cylindrical symmetry was broken by photoselection, i. e., by f lash excitation with low intensity linearly polarized light. From the measured electric signals, the threedimensional motion of the electric charge center in the bacteriorhodopsin molecules was calculated for the first 400 s. Simultaneous absorption kinetic recording provided the time-dependent concentrations of the intermediates. Combining the two sets of data, we determined the discrete dipole moments of intermediates up to M. When compared with the results of current molecular dynamics calculations, the data provided a decisive experimental test for selecting the optimal theoretical model for the proton transport and should eventually lead to a full description of the mechanism of the bacteriorhodopsin proton pump.Bacteriorhodopsin (bR) is an integral protein in the plasma membrane of Halobacterium salinarum (1). Upon light absorption, bR transports protons across the membrane, converting the photon energy into the energy of a proton electrochemical gradient (for recent reviews, see refs. 2 and 3). bR is a single small protein and is the simplest known active ion pump and biological light energy transducer. Consequently, it is a prototype system for studying the basic steps and rules of biological energy transduction. bR consists of the 23-kDa protein moiety and a retinal chromophore attached to the side chain of Lys-216 via a protonated Schiff base. The retinal is located about halfway through the membrane and sustains an angle of Ϸ70°with the membrane normal. Trimers of bR form twodimensional crystalline patches in the plasma membrane. These purple membrane fragments (pm) can be isolated as sheets of about 0.5-m diameter.

A Schiff base connectivity switch in sensory rhodopsin signaling

“…Integration of the current over time yields the overall charge displacement (28). The positive (outwardly directed toward the extracellular side) charge movement correlates with the fast component of Schiff base deprotonation as measured by accumulation of the blue-shifted M (also called S373) photointermediate in the same sample.…”

Target Analysis of the Bacteriorhodopsin Photocycle Using a Spectrotemporal Model

“…It is important to distinguish the way that the spectral information is dealt with. On one hand, the spectra can be derived from certain (sometimes arbitrary) assumptions, ,,,− and the accompanying concentration profiles are subsequently analyzed with a kinetic model. , On the other hand, the difference absorption spectra can be free parameters to be estimated simultaneously with the kinetic, thermodynamic, and model parameters. ,, The latter approach is much more complicated but less arbitrary because it is not necessary to choose the spectra of the intermediates in advance. The estimated difference absorption spectra also provide a test for the physicochemical relevance of the model used.…”

Photocurrents Generated by Bacteriorhodopsin Adsorbed on Thiol/Lipid Bilayers Supported by Mercury

“…first reported the PM to be adsorbed on black lipid membranes (BLMs) with the EC side turned preferentially toward the lipid film. Photocurrents and photovoltages generated by bR were investigated on BLMs, − on solid electrodes used as supports, , on oriented multilayer assemblies, in a polyacrylamide gel, − and on alkanethiol/phospholipid bilayers supported by a gold electrode . In these studies, the pH − and voltage dependence of the electrogenic events in the bR photocycle were investigated on both wild-type bR and mutants.…”