Photoelectric Measurements of Membranes

Light-induced HI release and reuptake as well as surface potential changes inherent in the bacteriorhodopsin reaction cycle were measured between 10'C and 5OC. Signals of optical pH indicators covalently bound to Lys-129 at the extracellular surface of bacteriorhodopsin were compared with absorbance changes of probes residing in the aqueous bulk phase. Only surface-bound indicators monitor the kinetics of H+ ejection from bacteriorhodopsin and allow the correlation of the photocycle with the pumping cycle.During the L5,50 -M412 transition the H+ appears at the extracellular surface of bacteriorhodopsin. Surface potential changes detected by bound fluorescei or by the potentiometric probe 4-{[2-(di-n-butylamino)-6-naphthyllvinyl}-1-(3-sulfopropyl)pyridinium betaine (di-4-ANEPPS) occur in milliseconds concomitantly with the formation and decay of the N intermediate. pH indicators residing in the aqueous bulk phase reflect the transfer of H+ from the membrane surface into the bulk but do not probe the early events of H+ pumping. The observed retardation of H+ at the membrane surface for several hundred microseconds is of relevance for energy conversion of biological membranes powered by electrocbemical H+ gradients.A challenge in membrane research is the understanding of generation, maintenance, and utilization of electrochemical H+ gradients. Especially the translocation of H+ across integral membrane proteins and subsequent H+ transfer into the aqueous bulk phase are less than well understood. Analysis of vectorial H+ transfer reactions is hampered by strong interactions of H+ with the membrane surface-i.e., with lipid head groups and exposed amino acids (1). These interactions will severely influence the rate of diffusioncontrolled transfer of H+ into the aqueous bulk phase (2). Therefore, it is necessary to monitor the kinetics of H+ transfer directly at the membrane surface, e.g., close to the H+ ejection site of an integral membrane protein (3).Bacteriorhodopsin (BR) in the purple membrane (PM) of Halobacterium halobium is a very attractive system for the investigation of molecular steps in light-driven vectorial H+ translocation. Although BR is an integral membrane protein (26,500 Da) with seven transmembrane a-helices, its structure is known at 3.5-to 7.8-A resolution, including the approximate position of many of the 248 amino acids (4). From the position of the chromophore retinal (5, 6), bound to Lys-216, the location of the protonated nitrogen of the Schiff base that is part of the active center of this transport protein can be inferred. To correlate H+ ejection kinetics with the corresponding spectroscopic intermediate(s) of the BR photocycle (7,8), with protonation changes of amino acids (9-12), and with alterations of the tertiary structure of the protein in the vicinity of the chromophore (13, 14), H+ transfer was monitored with the optical pH indicator fluorescein covalently linked to the extracellular surface of BR, at Lys-129. Concomitant surface potential changes were reflected by absor...

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“…4A). However, in electrical measurements a component in the same time domain had often been observed (29).…”

Section: Resultsmentioning

confidence: 99%

Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.

Heberle

Dencher

1992

Proc. Natl. Acad. Sci. U.S.A.

162

164

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“…Fast electrical signals with similar properties have been recorded from archaeal and visual rhodopsins, in which they derive from intramolecular charge movements triggered by photoexcitation (Trissl, 1990;Oroszi et al, 2002;Hong, 2004). Calculations, however, show that the amplitude of such displacement signals in Chlamydomonas cells, which contain relatively low concentrations of receptors, would probably be below the detection limit.…”

Section: Molecular Mechanism Of Actionmentioning

confidence: 99%

Sensory Rhodopsin Signaling in Green Flagellate Algae

Sineshchekov¹,

Spudich²

2005

Handbook of Photosensory Receptors

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“…This is due to the highly directional nature of this membrane protein, in which membrane orientation determines the polarity of the photoelectric response. [9,10] Among the methods used so far for membrane immobilization, we note Langmuir±Blodgett (LB) deposition, [11±14] electrostatic (layer-by-layer) self-assembly, [8,15] molecular recognition, [9,16] adsorption onto a lipid monolayer, [17] and sedimentation under a direct-current (DC) bias [18] or under the combined action of electric and magnetic fields. [19] In addition to the orientation, the thickness of bR films should also be controlled for the fabrication of bR-based bio-optoelectronic devices, especially if the nanometer-sized dimensions of the membranes are to be exploited.…”

mentioning

confidence: 99%