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1.
Subramaniam V Alves ID Salgado GF Lau PW Wysocki RJ Salamon Z Tollin G Hruby VJ Brown MF Saavedra SS 《Journal of the American Chemical Society》2005,127(15):5320-5321
Transmembrane proteins (TMPs), particularly ion channels and receptors, play key roles in transport and signal transduction. Many of these proteins are pharmacologically important and therefore targets for drug discovery. TMPs can be reconstituted in planar-supported lipid bilayers (PSLBs), which has led to development of TMP-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects TMP structure and activity. In this study, we have examined the effects of UV polymerization of bis-Sorbylphosphatidylcholine (bis-SorbPC) on the photoactivation of reconstituted bovine rhodopsin (Rho), a model G-protein-coupled receptor. Plasmon-waveguide resonance spectroscopy (PWR) was used to compare the degree of Rho incorporation and activation in fluid and poly(lipid) PSLBs. The results show that reconstitution of Rho into a supported lipid bilayer composed only of bis-SorbPC, followed by photoinduced lipid cross-linking, does not measurably diminish protein function. 相似文献
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Abstract— The phototactic response of Euglena gracilis is suppressed when the organisms are exposed to bright light of wavelength less than 650 nm. Activity returns in the dark, approximately 40 min being required for full restoration. Action spectral measurements demonstrate that the receptor pigment which mediates the suppression response may be a flavoprotein. Evidence is presented which indicates that photosuppression involves a direct action on the photo-tactic apparatus, perhaps a photobleaching of the tactic photoreceptor pigment. 相似文献
4.
Abstract— Chlorophyll, pheophytin and bacteriochlorophyll sensitize a one-electron transfer in the presence of quinones in ethanol, to produce a ternary complex of ground state porphyrin analog, alcohol cation radical and semiquinone anion radical as the primary photo-product at low temperature. A similar photo-oxidation of alcohol to produce a binary complex is caused by direct excitation of quin-one in the absence of chlorophyll. The mechanisms of these reactions and their implications for photosynthesis are discussed. 相似文献
5.
Laser flash photolysis was used to determine the kinetics of electron transfer between membrane-bound triplet chlorophyll (3C), cytochrome c (cyt c) located in the external water phase, and vesicle-reconstituted cytochrome c oxidase (CCO). 2,5-Di-t-butyl benzoquinone (2,5 TBQ) was used as an electron transfer mediator between 3C and cyt c. A light-induced cyclic electron transfer sequence between the redox components was observed (3C----2.5 TBQ----cyt c----CCO----C+.). Under optimum conditions of membrane surface charge and ionic strength, the overall efficiency of CCO reduction (based on 3C generated by the laser flash) was 14%. Under the anaerobic conditions used, CCO reoxidation (occurring via electron transfer to C+.) was quite slow (halftime approx. 1 s at 75 mM ionic strength). The multicomponent system displayed a high level of stability, as indicated by its ability to undergo many cycles of reduction and reoxidation without any apparent degradation of the components. These results demonstrate the feasibility of constructing complex electron transfer chains, including both soluble and membrane-bound redox proteins, in artificial lipid bilayers, whose properties can be readily controlled by manipulating parameters such as ionic strength and membrane composition. 相似文献
6.
Negatively charged vesicle suspensions containing chlorophyll a (chl) dissolved in the lipid bilayer, flavin mononucleotide (FMN) and/or ethylenediaminetetraacetic acid (EDTA) enclosed in the inner compartment as electron sources and oxidized cytochrome c (cyt c[ox]) in the outer compartment as an electron acceptor have been studied using laser flash photolysis and steady-state irradiation methods. Cytochrome c initially quenches the chl triplet state (3chl) generating the chlorophyll cation radical (chi+′) in the membrane. Reverse electron transfer from cyt c(red) to chl+. subsequently occurs in a kinetically biphasic reaction, with rate constants of 430 pT 30 and 21.9 pT 1.7 s?1 for the fast and slow phases, respectively. In the absence of FMN, reduction of chl+′ by EDTA in the inner compartment can be observed during steady-state irradiation but not in a laser flash photolysis experiment. This is due to a low reaction yield, which is probably limited by the repulsive electrostatic interaction between EDTA and the negatively charged membrane. When FMN was enclosed together with EDTA in the inner Compartment, the reaction yield of vectorial electron transfer across the bilayer from EDTA to cyt c(oX) was increased by a factor of six during steadystate white light irradiation. Laser flash photolysis and steady-state irradiation experiments using red and blue light excitation have demonstrated that the enhancement mechanism involves the formation of fully reduced FMN by blue light-sensitized photooxidation of EDTA via the flavin triplet state, occumng simultaneously with red lightsensitized electron transfer to cyt c via the chlorophyll triplet state. 相似文献
7.
Abstract— We have determined the chlorophyll triplet quenching efficiencies, the chlorophyll cation radical yields and the conversion efficiencies of chlorophyll triplet to radical in large and small unilamellar phosphatidylcholine vesicles (LUV and SUV, respectively) in the presence of electrically-charged electron acceptors (ferricyanide and oxidized cytochrome c) located in either the inner or outer aqueous compartments of the vesicles. Both types of vesicles displayed inside-outside asymmetry, although the properties were reversed. Triplet quenching in SUV was more efficient when ferricyanide was located within the vesicle interior, whereas the reverse was true in LUV. When ferricyanide was located on the outside of the vesicles, the extent of triplet quenching in LUV was about two times that in SUV and the amount of cation radical formed in LUV was about two times that in SUV. Under these conditions, the conversion efficiencies of chlorophyll triplet to radical were 12.2% for LUV and 8.5% for SUV. With cytochrome c as an electron acceptor in negatively charged vesicles (25 mol per cent dixhexadecylphosphate incorporated) similar results were obtained. Again, the triplet quenching and radical yield inside-outside asymmetry properties were reversed between the two types of vesicles, and radical formation efficiencies when cyt c was located outside the vesicles were higher in LUV (11.7%) than in SUV (4.2%). We conclude that the inside-outside asymmetric photochemical behavior of unilamellar phosphatidylcholine vesicles is influenced by factors in addition to the difference in radius of curvature between the inside and outside surfaces. It is suggested that transmembrane electrostatic potentials may be involved. Furthermore, in the present system the properties of LUV were more favorable to photochemical electron transfer product formation than those of SUV. 相似文献
8.
Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from P700 in Photosystem I (PSI)-enriched particles from spinach to the ferredoxins from spinach and the green alga Monoraphidium braunii. Very similar behavior for the interaction of both ferredoxins with the PSI complex was observed, although the algal ferredoxin appears to be slightly more effective as an electron acceptor. For both proteins, a non-linear protein concentration dependence of the rate constant for reduction was obtained, indicating complex formation preceding electron transfer. Estimates of 3 times 107M?1 s?1 and 140–180 s?l were obtained from these data for the second order rate constants for complex formation, and the limiting first order rate constants for electron transfer, respectively. At neutral pH, a biphasic dependence of the rate constant for ferredoxin reduction on the concentration of NaCl or MgCl2 was observed. This was interpreted in terms of the electrostatic interactions which occur between ferredoxin and the PSI membrane. In addition, magnesium cations appear to play a specific role in the interaction between PSI and ferredoxin. Thus, the addition of these ions under optimal conditions induces a 6-f-old increase in the electron transfer reaction rate constant, compared with a 2-f-old increase in the presence of an optimal amount of NaCI. This cannot be explained as arising from ionic strength effects. To our knowledge, this is the first time that a direct measurement of the rate constant for the reduction of ferredoxin by the PSI complex has been reported. 相似文献
9.
Chlorophyll-a was incorporated into cellulose acetate films and the triplet state decay kinetics and electron transfer from triplet to p-benzoquinone in aqueous solution was studied using laser flash photolysis and EPR. The triplet was found to decay by first order kinetics with a rate constant which was independent of Chl concentration. The triplet yield, however, was concentration dependent. These properties are due to quenching which occurs only at the singlet state level. In the presence of quinone, the triplet is quenched and, when the quinone is in an aqueous solution in contact with the film, Chl cation radical (C±) as well as the semiquinone anion radical (Q±) can be observed. The C decays by second order kinetics with a rate constant of 1.5 × 106M-1 s-1. Although triplet conversion to radicals is slightly lower in the films as compared to fluid solutions (? 3 times), the lifetimes of the radicals are greatly increased (? 103 times). 相似文献
10.
Abstract— The characterization and kinetic analysis by laser Rash photolysis of an improved model system for observing chlorophyll a photosensitized electron transfer across a lipid bilayer membrane is described. In this system, the electron acceptor is a water-soluble naphthoquinone, S-(2-methyl-l,4-naphthoquinonyl-3)-glutathione (MGNQ) which is dissolved in the inner aqueous compartments of phospholipid bilayer vesicles, and the electron donor is glutathione (GSH) which is dissolved in the outer aqueous phase. Chlorophyll (Chl) is dissolved in the membrane. Oxidative quenching of the triplet state of Chl by the quinone at the inner surface of the vesicle produces the Chl+ and MGNQ- radicals. Chi+ is reduced by GSH at the outer surface of the vesicle (k= 2.6 × 106M-1 s-1) in competition with the recombination between Chl+. and MGNO- (k= 2.5 × 103 S-1). It is shown that a kinetic mechanism involving competition between recombination, electron transfer across the bilayer, and reduction by donor at the opposite surface can quantitatively account for the decay of Chl+. Electron transport across the bilayer is postulated to occur by a two-step mechanism involving electron exchange between Chl and Chl+ within the lipid monolayer (k= 3.2 × 106 M-1 s-1) and across the bilayer. The rate constant for the latter exchange process approaches 104 s-1 as the concentration of Chl in the bilayer increases. Under appropriate conditions, approximately 20% of all photons absorbed by the vesicle system result in electron transfer across the mcmbrane from GSH to MGNQ. 相似文献