The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions

The interrelation was studied between the phototransient absorbing maximally at 412 nm (M₄₁₂) and light-induced proton release under steady-state conditions in aqueous suspensions of ‘purple membrane’ derived from Halobacterium halobium. The decay of M₄₁₂ was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M₄₁₂ which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M₄₁₂ was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M₄₁₂ without affecting the level of M₄₁₂ which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca²⁺ and their antagonists La³⁺ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

On the two forms of bacteriorhodopsin

In our previous work [(1993) FEBS Lett. 313, 248-250; (1993) Biochem. Int. 30,461-469] M-intermediate formation of wild-type bacteriorhodopsin was shown to involve two components differing in time constants (τ₁ = 60–70 μs and τ₂ = 220–250 μs), which were suggested to reflect two independent pathways of M-intermediate formation. The contribution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the D115N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:1. Upon lowering the pH, the slow phase of M-formation vanished at a pK of 6.2, and in the pH region 3.0–5.5 only the M-intermediate with a rise time of 60 μs was present. A 5–6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M-intermediate formation. The remaining bacteriorhodopsin contained 100% fast M-intermediate. The disappearance of the 250-μs phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.     

Molecular dynamics simulations of photoactive yellow protein (PYP) in three states of its photocycle: a comparison with X-ray and NMR data and analysis of the effects of Glu46 deprotonation and mutation

Photoactive yellow protein (PYP) is a prototype of the PAS domain superfamily of signaling proteins. The signaling process is coupled to a three-state photocycle. After the photoinduced trans-cis isomerization of the chromophore, 4-hydroxycinnamic acid (pCA), an early intermediate (pR) is formed, which proceeds to a second intermediate state (pB) on a sub-millisecond time scale. The signaling process is thought to be connected to the conformational changes upon the formation of pB and its recovery to the ground state (pG), but the exact signaling mechanism is not known. Experimental studies of PYP by solution NMR and X-ray crystallography suggest a very flexible protein backbone in the ground as well as in the signaling state. The relaxation from the pR to the pB state is accompanied by the protonation of the chromophore's phenoxyl group. This was found to be of crucial importance for the relaxation process. With the goal of gaining a better understanding of these experimental observations on an atomistic level, we performed five MD simulations on the three different states of PYP: a 1 ns simulation of PYP in its ground state [pG(MD)], a 1 ns simulation of the pR state [pR(MD)], a 2 ns simulation of the pR state with the chromophore protonated (pRprot), a 2 ns simulation of the pR state with Glu46 exchanged by Gln (pRGln) and a 2 ns simulation of PYP in its signaling state [pB(MD)]. Comparison of the pG simulation results with X-ray and NMR data, and with the results obtained for the pB simulation, confirmed the experimental observations of a rather flexible protein backbone and conformational changes during the recovery of the pG from the pB state. The conformational changes in the region around the chromophore pocket in the pR state were found to be crucially dependent on the strength of the Glu46-pCA hydrogen bond, which restricts the mobility of the chromophore in its unprotonated form considerably. Both the mutation of Glu46 with Gln and the protonation of the chromophore weaken this hydrogen bond, leading to an increased mobility of pCA and large structural changes in its surroundings. These changes, however, differ considerably during the pRGln and pRprot simulations, providing an atomistic explanation for the enhancement of the rate constant in the Gln46 mutant. Electronic supplementary material to this article is available at and is accessible for athorized users. Electronic Publication     

Time-resolved quasielastic neutron scattering studies of native photosystems

The internal molecular dynamics of proteins plays an important role in a number of functional processes in native photosystems. Prominent examples include the photocycle of bacteriorhodopsin and electron transfer in the reaction center of plant photosystem II. In this regard, the recently developed technique of time-resolved quasielastic neutron scattering with laser excitation opens up new perspectives for the study of protein/membrane dynamics in specific functional states of even complex systems. The first direct observation of a functionally modulated protein dynamics has just recently been reported for the model system bacteriorhodopsin (Pieper et al., Phys. Rev. Lett. 100, 2008, 228103.), where a transient softening of the protein was observed on a timescale of ∼ 1 ms along with the large-scale structural change in the M-intermediate of bacteriorhodopsin. In contrast, photosystem II membrane fragments with inhibited electron transfer show a suppression of protein dynamics ∼160 μs after the actinic laser flash (Pieper and Renger, Biochemistry 48, 2009, 6111). This effect may reflect aggregation-like conformational changes capable of dissipation of excess excitation energy to prevent photodamage in the absence of Q_A→Q_B electron transfer. These findings indicate that proteins exhibit a remarkable flexibility to accommodate different functional processes. This contribution will discuss methodical aspects, challenges, and recent applications of laser-excited, time-resolved quasielastic neutron scattering.     

The pH-dependence of photochemical intermediates of O and P in bacteriorhodopsin by continuous light

The pH-dependence of the O and P intermediates in the photocycle of bacteriorhodopsin (bR) on the intensity and duration of the exciting flash was investigated for bR glycerol suspensions and bR gelatin films. Green and red laser flashes (532 and 670 nm) were utilized to generate a photoequilibrium state of bR and O at ambient temperature, and UV-vis spectroscopy was used to determine the photoconversion for the bR suspensions and films. The maximal concentration of the O intermediate was observed to be pH-dependent and the dependency was most pronounced at a slightly alkaline pH values. The photochemical conversion from the O to P intermediate was investigated for both bR suspensions and films. The P intermediate was only found in bR gelatin film. These results indicate that bR gelatin film may be an attractive candidate for the information storage based on P intermediate. It is possible, with red light, to create photoproducts which are thermally stable at ambient temperature and that can be photochemically erased.     

Variance reduction by simultaneous multi-exponential analysis of data sets from different experiments

The analysis of experimental data from the photocycle of bacteriorhodopsin (bR) as sums of exponentials has accumulated a large amount of information on its kinetics which is still controversial. One reason for ambiguous results can be found in the inherent instabilities connected with the fitting of noisy data by sums of exponentials. Nevertheless, there are strategies to optimize the experiments and the data analysis by a proper combination of well known techniques. This paper describes an applicable approach based on the correct weighting of the data, a separation of the linear and the non-linear parameters in the process of the least squares approximation, and a statistical analysis applying the correlation matrix, the determinant of Fisher's information matrix, and the variance of the parameters as a measure of the reliability of the results. In addition, the confidence regions for the linear approximation of the non-linear model are compared with confidence regions for the true non-linear model. Evaluation techniques and rules for an optimum experimental design are mainly exemplified by the analysis of numerically generated model data with increasing complexity. The estimation of the number of exponentials significant for the interpretation of a given set of data is demonstrated by using records from eight absorption and photocurrent experiments on the photocycle of bacteriorhodopsin. Offprint requests to: K.-H. Müller     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Detection and expression of a gene encoding a new bacteriorhodopsin from an extreme halophile strain HT (JCM 9743) which does not possess bacteriorhodopsin activity

Membrane vesicles prepared from an extreme halophile strain, HT (JCM 9743), showed no bacteriorhodopsin activity. However, a DNA fragment, amplified by polymerase chain reaction (PCR), appeared to encode the C to G helices of a bacteriorhodopsin(bR)-like protein. With the PCR product as a probe, the gene coding for a novel bacteriorhodopsin was cloned from the genomic DNA of the strain HT. The open reading frame of the gene was ligated with the promoter region of the bop gene of Halobacterium salinarum bR, and expressed in a bR-deficient host strain, L33, using the plasmid vector pXLNov-R. The purplish membrane fraction purified from cells of a transformant exhibited a cyclic photoreaction characteristic of bacteriorhodopsin. Received: August 12, 1997 / Accepted: October 20, 1997     

The reaction cycle of bacteriorhodopsin: an analysis using visible absorption,photocurrent and infrared techniques

The light activated absorbance changes and photo-electric events of bacteriorhodopsin (bR) were simultaneously measured. The results were compared with the kinetics of the time resolved infrared signals which are characteristic for protonation changes of Asp residues, chromophore vibrations, and amide I vibrations. Each data set was analyzed separately. Assuming first order reactions the experimental curves in the time range from L back to bR could be fitted by a sum of five exponentials. However, for the photocurrent signal only four exponentials were necessary. The corresponding half-life times were of the same order of magnitude. Simultaneous fits of the traces from absorption changes in the visible range and the photocurrent signal provided evidence that the photocurrent data could also be described by the same sum of exponentials as the data obtained in the visible range. The rate constants obtained from the different methods applied were, within the limits of error, identical. These results demonstrate that retinal monitors not only charge displacements but also conformational movements of the protein moiety. The reprotonation of the Schiff base occurs synchronously with a protonation change of an internal aspartic acid which absorbs at 1755 cm^–1. From the IR-signals, amplitude spectra could be derived which provided evidence that Asp-residues absorbing at 1765 cm^–1 (Asp85) and 1755 cm^–1 are still protonated in the O-intermediate. Major conformational changes of the peptide back bone occur in the time range of the L M transition and with opposite sign during the decay of the O-intermediate. Offprint requests to: M. Engelhard