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1.
The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy. 相似文献
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V I Tsetlin T N Alyonycheva V V Shemyakin L A Neiman V T Ivanov 《European journal of biochemistry》1988,178(1):123-129
The action of thermally activated tritium on the purple membrane and delipidated bacteriorhodopsin fragments has been studied, tritium incorporation into specified amino acid residues being quantified by Edman degradation. The membrane environment was found to affect the accessibility of amino acid residues for tritium. Bacteriorhodopsin fragments 14-31, 45-63, 81-89, 171-179, and 210-225 were localized to the membrane interior while fragments 4-12, 32-44, 64-65, 73-80, and 156-170 should lie outside or close to membrane surface. It was demonstrated that the peptide fragments joining transmembrane rods are not fully exposed to the solution. 相似文献
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We report on a differential scanning calorimetry study of native purple membranes under the following solvent conditions: 50 mM carbonate-bicarbonate, 100 mM NaCl, pH 9.5 and 190 mM phosphate, pH 7.5. The calorimetric transitions for bacteriorhodopsin denaturation are highly scanning-rate dependent, which indicates that the thermal denaturation is under kinetic control. This result is confirmed by a spectrophotometric study on the kinetics of the thermal denaturation of this protein. The calorimetric data at pH 9.5 conform to the two-state irreversible model. Comments are made regarding the information obtainable from differential scanning calorimetry studies on bacteriorhodopsin denaturation and the effect of irreversibility on the stability of membrane proteins.
Correspondence to: J. M. Sanchez-Ruiz 相似文献
4.
Generation of electric potential difference by bacteriorhodopsin proteoliposomes incorporated into the phospholipid-impregnated collodion film has been studied. It is shown that illumination of this film by continuous light gives rise to the generation of an electric potential difference across the film (plus on the bacteriorhodopsin-free side), which can be as high as 300 mV. Short unsaturating flash inducing single turn-over of bacteriorhodopsin generates the potential difference which is a function of the flash intensity (70 mV at 3 mjoule light). The flash-induced photoelectric response consists of four phases. (1) Very fast (tau less than 1 microsec) generation of a potential difference (minus in the bacteriorhodopsin-free compartment). The amplitude of this phase is rather small (1--5 mV). (2) Fast phase of positive charging of the bacteriorhodopsin-free compartment (tau = 25--50 microsec). (3) Slow phase of positive charging of this compartment (tau = 6--12 msec) Amplitude of the second phase is to that of the third as 1 : 2. (4) A very slow phase of discharge of the flash-induced potential difference (tau = 1 sec at 10(8) ohm X cm2 film resistance). The third phase was specifically inhibited by La3+. Both the second and the third phases are decelerated by substitution of D2O in 4.5--5 and 2 times, respectively, while the amplitude of the first phase increases. Prolonged storage of the system in the dark (tua = 20--25 min) causes the decrease in the amplitudes of the second and the third phases as if the amount of active bacteriorhodopsin molecules were increased by factor 2. Such an inhibition was reversed by 30--60 sec illumination of the system. The dark adaptation is accompanied by some increase in the first phase amplitude. Comparison of these data with results of other studies on bacteriorhodopsin suggests that (1) the first phase is due to the photoinduced change in the retinal dipole; (2) the second phase corresponds to H+ transfer from Schiff base to the water solution in the proteoliposome interior; 3) the third phase represents H+ transfer from the incubation mixture to Schiff base; (4) the dark adaptation is a result of transition of photoelectrochemically active all-trans-retinal to the inactive 13-cis-retinal. 相似文献
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Correlation between absorption maxima and thermal isomerization rates in bacteriorhodopsin 总被引:2,自引:2,他引:0
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Steven J. Milder 《Biophysical journal》1991,60(2):440-446
The reported rates of thermal 13-cis to all-trans isomerization of the protonated Schiff base of retinal (PSBR) in solution and in bacteriorhodopsin (BR) are shown to be correlated with the red shift in the absorption maximum of the chromophore, though the linear fit is different for BR and for a model PSBR in solution. Because the red shift in the absorption has been previously shown to be correlated with π-electron delocalization in the chromophore, this suggests that the thermal isomerization rate is largely regulated by the amount of double bond character in the chromophore. Because the linear fit of isomerization rates with absorption maxima is different for BR and the model PSBR, specific interactions of the protein with the chromophore must also be a factor in determining thermal isomerization rates in BR. A model of the later steps in the photocycle of BR is presented in which the 13-cis to all-trans thermal isomerization occurs during the O intermediate. 相似文献
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The bacteriorhodopsin model membrane system as a prototype molecular computing element 总被引:2,自引:0,他引:2
F T Hong 《Bio Systems》1986,19(3):223-236
The quest for more sophisticated integrated circuits to overcome the limitation of currently available silicon integrated circuits has led to the proposal of using biological molecules as computational elements by computer scientists and engineers. While the theoretical aspect of this possibility has been pursued by computer scientists, the research and development of experimental prototypes have not been pursued with an equal intensity. In this survey, we make an attempt to examine model membrane systems that incorporate the protein pigment bacteriorhodopsin which is found in Halobacterium halobium. This system was chosen for several reasons. The pigment/membrane system is sufficiently simple and stable for rigorous quantitative study, yet at the same time sufficiently complex in molecular structure to permit alteration of this structure in an attempt to manipulate the photosignal. Several methods of forming the pigment/membrane assembly are described and the potential application to biochip design is discussed. Experimental data using these membranes and measured by a tunable voltage clamp method are presented along with a theoretical analysis based on the Gouy-Chapman diffuse double layer theory to illustrate the usefulness of this approach. It is shown that detailed layouts of the pigment/membrane assembly as well as external loading conditions can modify the time course of the photosignal in a predictable manner. Some problems that may arise in the actual implementation and manufacturing, as well as the use of existing technology in protein chemistry, immunology, and recombinant DNA technology are discussed. 相似文献
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Structure and thermal stability of monomeric bacteriorhodopsin in mixed phospholipid/detergent micelles 总被引:3,自引:0,他引:3
Thermal unfolding experiments on bacteriorhodopsin in mixed phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 +/- 13 A in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Proton uptake mechanism of bacteriorhodopsin as determined by time-resolved stroboscopic-FTIR-spectroscopy 总被引:5,自引:5,他引:0
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Bacteriorhodopsin's proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 μs, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k7 = 0.3 ms, k4 = 2.3 ms, k3 = 6.9 ms, k6 = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k4), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M1 to M2 transition. However, experimentally two different M intermediates are not resolved, and M2 and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M2) transition. This reaction is proposed to be involved in the “reset switch” of the pump, (b) In the M to N (conceptionally M2) transition, most likely, asp-85's carbonyl frequency shifts from 1,762 to 1,753 cm-1 and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O. 相似文献
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Upon addition of hydroxylamine to chloroplasts or photosystem II preparations, the EPR signal of Z? disappears and a new signal is observed. From its shape and g-value this signal is identified with the oxidized reaction center chlorophyll, P680+. The decay of P680+ occurs with a halftime of ? 200 μs and apparently is the result of a back reaction with the reduced form of the primary acceptor, QA. This mode of hydroxylamine inhibition is reversible. These observations indicate that hydroxylamine, in addition to its well known inhibitory action on the oxygen evolving complex, is also able to disrupt physiological electron flow to P680 itself. 相似文献
14.
Fourier transform infrared study of the effect of different cations on bacteriorhodopsin protein thermal stability 总被引:2,自引:0,他引:2
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The effect of divalent ion binding to deionized bacteriorhodopsin (dI-bR) on the thermal transitions of the protein secondary structure have been studied by using temperature-dependent Fourier transform infrared (FT-IR) spectroscopy. The native metal ions in bR, Ca(2+), and Mg(2+), which we studied previously, are compared with Mn(2+), Hg(2+), and a large, synthesized divalent organic cation, ((Et)(3)N)(2)Bu(2+). It was found that in all cases of ion regeneration, there is a pre-melting, reversible conformational transition in which the amide frequency shifts from 1665 to 1652 cm(-1). This always occurs at approximately 80 degrees C, independent of which cation is used for the regeneration. The irreversible thermal transition (melting), monitored by the appearance of the band at 1623 cm(-1), is found to occur at a lower temperature than that for the native bR but higher than that for acid blue bR in all cases. However, the temperature for this transition is dependent on the identity of the cation. Furthermore, it is shown that the mechanism of melting of the organic cation regenerated bR is different than for the metal cations, suggesting a difference in the type of binding to the protein (either to different sites or different binding to the same site). These results are used to propose specific direct binding mechanisms of the ions to the protein of deionized bR. 相似文献
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Structural features on the extracellular side of the D85S mutant of bacteriorhodopsin (bR) suggest that wild-type bR could be a hydroxyl-ion pump. A position between the protonated Schiff base and residue 85 serves as an anion-binding site in the mutant protein, and hydroxyl ions should have access to this site during the O-intermediate of the wild-type bR photocycle. The guanidinium group of R82 is proposed (1) to serve as a shuttle that eliminates the Born energy penalty for entry of an anion into this binding pocket, and conversely, (2) to block the exit of a proton or a related proton carrier. 相似文献
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Walther Stoeckenius Richard H. Lozier Werner Niederberger 《European biophysics journal : EBJ》1977,3(1):65-68
Bacteriorhodopsin is a membrane-bound light energy transducer which generates an electrochemical proton gradient. It undergoes a cyclic photoreaction in which five intermediates have been identified. During the cycle it releases a proton from one surface of the membrane and takes up a proton on the opposite surface. The active chromophore consists of retinal bound through a Schiff base to the protein. The Schiff base is deprotonized during the photoreaction cycle and appears to be involved in the transport of protons through the membrane. The retinal may also undergo an isomerization.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976This work was supported by NASA Grant NSG-7151 and NHLI Grant HL-06285 相似文献
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Reconstitution of bacteriorhodopsin 总被引:10,自引:0,他引:10
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To determine the strength of noncovalent interactions that stabilize a membrane protein complex, we have developed an in vitro method for quantifying the dissociation of the bacteriorhodopsin (BR) lattice, a naturally occurring two-dimensional crystal. A lattice suspension was titrated with a short- and long-chain phosphatidylcholine mixture to dilute BR within the lipid bilayer. The fraction of BR in the lattice form as a function of added lipid was determined by visible circular dichroism spectroscopy and fit with a cooperative self-assembly model to obtain a critical concentration for lattice assembly. Critical concentration values of wild-type and mutant proteins were used to calculate the change in lattice stability upon mutation (DeltaDeltaG). By using this method, a series of mutant proteins was examined in which residues at the BR-BR interface were replaced with smaller amino acids, either Ala or Gly. Most of the mutant lattices were destabilized, with DeltaDeltaG values of 0.2-1.1 kcal/mol at 30 degrees C, consistent with favorable packing of apolar residues in the membrane. One mutant, I45A, was stabilized by approximately 1.0 kcal/mol, possibly due to increased lipid entropy. The DeltaDeltaG values agreed well with previous in vivo measurements, except in the case of I45A. The ability to measure the change in stability of mutant protein complexes in a lipid bilayer may provide a means of determining the contributions of specific protein-protein and protein-lipid interactions to membrane protein structure. 相似文献