Light-driven Proton Pump Bacteriorhodopsin Research Articles

Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing

The topography of membrane-surface-exposed amino acids in the light-driven proton pump bacteriorhodopsin (BR) was studied. By limited proteolysis of purple membrane with papain or proteinase K, domains were cleaved, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Fragments transferred were sequenced in a gas-phase sequencer. Papain cleavage sites at Gly-65, Gly-72, and Gly-231, previously only deduced from the apparent molecular weight of the digestion fragments, could be confirmed by N-terminal micro-sequencing. By proteinase K, cleavage occurred at Gln-3, Phe-71, Gly-72, Tyr-131, Tyr-133, and Ser-226, i.e., in regions previously suggested to be surface-exposed. Additionally, proteinase-K cleavage sites at Thr-121 and Leu-127 were identified, which are sites predicted to be in the α-helical membrane-spanning segment D. Our results, especially that the amino acids Gly-122 to Tyr-133 are protruding into the aqueous environment, place new constraints on the amino-acid folding of BR across the purple membrane. The validity of theoretical prediction methods of the secondary structure and polypeptide folding for membrane proteins is challenged. The results on BR show that micro-sequencing of peptides separated by SDS-PAGE and blotted to PVDF can be successfully applied to the study of membrane proteins.

Intramolekulare Proteindynamik untersucht mit zeitaufgelöster Fourier Transform Infrarot‐Differenzspektroskopie

AbstractThe intramolecular reactions of membrane proteins are investigated by the new developed time resolved FTIR difference spectroscopy technique. By this technique the kinetics of single groups in membrane proteins can be determined. As example the protonation kinetics of aspartic acids are shown. Simultaneously the absorbance change in the visible spectral region is measured at one wavelength. Thereby the IR‐Absorbance changes can be correlated to changes in the visible spectral region. The technique is applied to the lightdriven proton pump bacteriorhodopsin and to crystals of the photosynthetic reaction center of Rps. viridis.

Direct observation of the femtosecond excited-state cis-trans isomerization in bacteriorhodopsin.

Femtosecond optical measurement techniques have been used to study the primary photoprocesses in the light-driven transmembrane proton pump bacteriorhodopsin. Light-adapted bacteriorhodopsin was excited with a 60-femtosecond pump pulse at 618 nanometers, and the transient absorption spectra from 560 to 710 nanometers were recorded from -50 to 1000 femtoseconds by means of 6-femtosecond probe pulses. By 60 femtoseconds, a broad transient hole appeared in the absorption spectrum whose amplitude remained constant for about 200 femtoseconds. Stimulated emission in the 660- to 710-nanometer region and excited-state absorption in the 560- to 580-nanometer region appeared promptly and then shifted and decayed from 0 to approximately 150 femtoseconds. These spectral features provide a direct observation of the 13-trans to 13-cis torsional isomerization of the retinal chromophore on the excited-state potential surface. Absorption due to the primary ground-state photoproduct J appears with a time constant of approximately 500 femtoseconds.

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin.

A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., & Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-100-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated.

Calcium transport in membrane vesicles of Streptococcus cremoris

Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A.J.M., Hellingwerf, K.J. & Konings, W.N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing delta psi with a concomitant increase in delta pH stimulates Ca2+ uptake while dissipation of the delta pH results in a reduced rate of Ca2+ uptake. Also an artificially generated delta pH, interior acid, can drive Ca2+ uptake in S. cremoris membrane vesicles. Ca2+ uptake depends strongly on the presence of external phosphate while Ca2+-efflux-induced proton flux is independent of the presence of external phosphate. Ca2+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,N'-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons.

Chromophore/protein interaction in bacterial sensory rhodopsin and bacteriorhodopsin

Retinal analogues with altered conjugated double bond systems or altered stereochemistry were incorporated into the phototaxis receptor sensory rhodopsin (SR) and the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium halobium. Wavelength shifts in absorption ("opsin shifts") due to analogue interaction with the protein microenvironment demonstrate that the same overall electrostatic and steric properties of the retinal binding-site structures exist in both proteins despite their different functions. pi-Electron calculations from the opsin shifts lead to a new description of protein charge distribution that applies to the binding sites of both SR and BR. The new data extends the previously proposed external point charge model for BR to include an ion-pair protein/chromophore interaction near the beta-ionone moiety. The new data modifies the previously proposed external point-charge model, the derivation of which involved an experimentally erroneous opsin shift for one of the BR analogues.

37] Generation of a protonmotive force in anaerobic bacteria by end-product efflux

Publisher Summary This chapter deals with the generation of a Protonmotive force in anaerobic bacteria by end-product efflux. The protonmotive force acts as a driving force or regulatory parameter in a large number of different metabolic processes. Until recently only three primary proton pumps have been described in bacteria: (1) membranebound electron transfer chains; (2) the proton-translocating adenosine triphosphatase (ATPase) complex; and (3) in halobacteria, the light-driven proton pump bacteriorhodopsin. A number of experimental approaches can supply information about the generation of metabolic energy by end-product efflux: (1) uptake experiments, to demonstrate carrier activity and to obtain information on the H + /end-product stoichiometry; (2) efflux experiments from preloaded membranes, to demonstrate end-product efflux driven Ap generation (and subsequent ATP synthesis); (3) simultaneous measurements of the end-product gradient and Ap in growing or actively metabolizing cells, to obtain information on the H + / end-product stoichiometry and possible contributions of energy recycling to the overall production of metabolic energy; and (4) yield experiments under various conditions.

Calcium transport in membrane vesicles ofStreptococcus cremoris

Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A. J. M.. Hellingwerf, K. J. & Konings, W. N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing AI~ with a concomitant increase in ApH stimulates Ca2+ uptake while dissipation of the ApH results in a reduced rate of Ca2+ uptake. Also an artificially generated ApH, interior acid, can drive CaZ+ uptake in S. cremoris membrane vesicles. CaZ+ uptake depends strongly on the presence of external phosphate while Ca'+-efflux-induced proton flux is independent of the presence of external phosphate. Caz+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,W-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons.

Light-induced generation of a protonmotive force and Ca 2+-transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes

The light-driven primary proton pump bacteriorhodopsin has been incorporated in the cytoplasmic membrane of Streptococcus cremoris, in order to generate a protonmotive force across these membranes. This has been achieved by fusion of S. cremoris membrane vesicles with bacteriorhodopsin proteoliposomes. This fusion occurred when both preparations were mixed at low pH (less than 6.0), as shown by sucrose density gradient centrifugation and by dilution of fluorescent phospholipids incorporated into the bacteriorhodopsin proteoliposomes. Fusion was strongly enhanced by the presence of negatively charged phospholipids in the liposomal bilayer. When proteoliposomes were used that showed light-dependent proton uptake, the orientation of bacteriorhodopsin in the fused membranes was inside-out with respect to the in vivo orientation in Halobacterium halobium. Consequently, in the light a ΔΨ, interior positive and a ΔpH, interior acid were generated. This protonmotive force could drive calcium uptake in the fused membranes. The uptake increased hyperbolically with increasing light intensity and was abolished by bleaching of bacteriorhodopsin. Addition of the ionophore valinomycin stimulated calcium uptake and led to an increase of the ΔpH. Calcium uptake was strongly decreased in the dark and in the light in the presence of uncouplers, nigericin or both valinomycin and nigericin.

Structure of the retinal chromophore in halorhodopsin: A resonance Raman study

Resonance Raman (RR) spectra of the light-driven chloride pump halorhodopsin (HR) were recorded after isolation of the protein from cell membranes of Halobacterium halobium. The spectra of the unphotolyzed state HR 578 were compared with those of two unphotolyzed species of the light-driven proton pump bacteriorhodopsin, BR 570 and BR 548, which were obtained from light- and dark-adapted purple membranes. Identical structural components in the retinal chromophores of HR 578 and BR 570 are found, both having the all- trans configuration of the retinal chain and a protonated Schiff base linkage to the protein with anti-position of the two hydrogens. Only minor conformational differences between the chromophoric structures in the two proteins could be inferred from the vibrational structure of the RR spectra. The function of the two chromophores as triggers of light-induced chloride or proton transport emphasizes the role of the protein part in the ion specificity of the pumps.

Simulations of frequency-dependent photoacoustic magnitude signals and their implications for bacteriorhodopsin photocycle energetics

The modulation frequency dependence of photoacoustic signals obtained from photoactive samples can provide information on the time-dependent enthalpy changes occurring during the light-induced process. The experimental requirements for this type of calorimetry, and the interpretation ot the results, are critically examined with reference to the light-driven proton pump bacteriorhodopsin. For a three-step unbranched model of the bacteriorhodopsin photocycle we derive an expression for the photoacoustic magnitude signal as a function of frequency. Simulations are performed for various values of the rate constants and energetic changes. It is shown that the net heat uptake during a low, final step postulated by some workers should be reflected in the photoacoustic magnitude frequency spectrum, giving rise to a characteristic maximum. However, this effect, which has been observed experimentally, may also be produced by a fast, strongly endothermic step occurring earlier. The precise chronology of an endothermic transition cannot be resolved unambiguously by magnitude measurements alone, although they are free from assumptions regarding difficult-to-measure phase relationships. Hence, the published photoacoustic observations showing the effect are consistent with a cyclic sequence of events in which the bacteriorhodopsin system first undergoes an increase of entropy, followed by a decrease on returning to the initial state, as well as the reverse. It is argued that the molecular disorder-order sequence is more probable.

Chapter 14 Three-Dimensional Reconstruction of Membrane Protein Crystals

This chapter describes the three-dimensional reconstruction of membrane protein crystals. Three-dimensional reconstruction has been applied to two-dimensional crystals of many biological molecules. Some membrane proteins are found to be crystalline in vivo . The halophilic bacterium Halobacterium halobium assembles its light-driven proton pump bacteriorhodopsin into well-ordered two-dimensional crystals, the purple membrane on the surface. Crystals can also be formed from protein purified by more standard methods and reconstituted. The two oxidase crystal forms have been prepared from purified beef heart enzyme. The purple membrane protein bacteriorhodopsin is segregated in crystalline patches on the surface of its bacterium. Reconstituted bacteriorhodopsin spontaneously forms crystals, and their order is unaffected by ionic strength and pH over a wide range. This suggests that the crystal-forming interaction is within the membrane. It is found that all the protein molecules in the crystal are arranged in trimers and have the same orientation. The Fourier transform can be used to calculate the average structure of a molecule in a crystal in a direct way.