Optical and electric signals from dried oriented purple membrane of bacteriorhodopsins.

All the intermediates of the bacteriorhodopsin photocycle are excitable with light of suitable wavelength. This property might regulate the activity in the cells when they are exposed in the nature to high light intensity. On the other hand this property is involved in many applications. In this study the ground state and M intermediate of dried oriented samples of wild-type bacteriorhodopsin and its mutant D96N were excited with 406 nm laser flashes. Substantial M populations were generated with quasi-continuous illumination. The decay of the absorption of M intermediate had three components: their lifetimes were very different for laser flash and quasi-continuous illuminations in cases of both bacteriorhodopsin species. The optical answer for the excitation of M intermediate had a lifetime of 2.2 ms. Electric signals for M excitation had large fast negative components and small positive components in the 100 µs time domain. The results are expected to have important implications for bioelectronic applications of bacteriorhodopsin.

Excitation of the L intermediate of bacteriorhodopsin: electric responses to test x-ray structures.

The L intermediate of bacteriorhodopsin was excited, and its electrical response was measured. Two positive components were found in it with respect to the direction of proton pumping: an unresolved fast component, and a slower one (tau=7 micros) of small amplitude. The fast component was assigned to a charge motion corresponding to reisomerization of the retinal moiety, whereas the slow one was attributed to charge rearrangements reestablishing the ground state. Because three x-ray crystallographic structures have recently been reported for the L intermediate, it seemed important to calculate the intramolecular dipole moment changes associated to bR-->L for all three structures, so as to compare them with similar quantities determined from the electrical signals. The results are discussed in terms of amino acid side chains possibly contributing to the observed effect. We propose to use electrical signals as a verification tool for intermediate structures of the photocycle, and thus for molecular models of proton pumping.

Actinic light-energy dependence of proton release from bacteriorhodopsin.

Measuring the light-density (fluence) dependence of proton release from flash excited bacteriorhodopsin with two independent methods we found that the lifetime of proton release increases and the proton pumping activity, defined as a number of protons per number of photocycle, decreases with increasing fluence. An interpretation of these results, based on bending of purple membrane and electrical interaction among the proton release groups of bacteriorhodopsin trimer, is presented.

Late events in the photocycle of bacteriorhodopsin mutant L93A.

In the photocycle of bacteriorhodopsin (bR) from Halobacterium salinarum mutant L93A, the O-intermediate accumulates and the cycling time is increased approximately 200 times. Nevertheless, under continuous illumination, the protein pumps protons at near wild-type rates. We excited the mutant L93A in purple membrane with single or triple laser flashes and quasicontinuous illumination, (i.e., light for a few seconds) and recorded proton release and uptake, electric signals, and absorbance changes. We found long-living, correlated, kinetic components in all three measurements, which-with exception of the absorbance changes-had not been seen in earlier investigations. At room temperature, the O-intermediate decays to bR in two transitions with rate constants of 350 and 1800 ms. Proton uptake from the cytoplasmic surface continues with similar kinetics until the bR state is reestablished. An analysis of the data from quasicontinuous illumination and multiple flash excitation led to the conclusion that acceleration of the photocycle in continuous light is due to excitation of the N-component in the fast N<-->O equilibrium, which is established at the beginning of the severe cycle slowdown. This conclusion was confirmed by an action spectrum.

Photoexcitation of the O-intermediate in bacteriorhodopsin mutant L93A.

During the extended lifetime of the O-state in bacteriorhodopsin (bR) mutant L93A, two substates have been distinguished. The first O-intermediate (OI) is in rapid equilibrium with N and apparently still has a 13-cis chromophore. OI undergoes a photoreaction with a small absorbance change, positive charge transport in the pumping direction, and proton release and uptake. None of these effects was detected after photoexcitation of the late O (OII). The most likely interpretation of the effects seen is an accelerated return of the molecule from the OI- to the bR-state. However, with a lifetime approximately 140 ms, the reaction cannot account for the observed high pumping efficiency of the mutant under continuous illumination. We suggest that OII corresponds to the O-intermediate with a twisted all-trans chromophore seen in the photocycle of wild-type bR, where the 13-cis OI-intermediate under the usual conditions does not accumulate in easily detectable amounts and, therefore, has generally been overlooked. Both the OI- and OII-decays are apparently strongly inhibited in the mutant.

Proton binding to biological membranes.

Biological membranes contain proton-binding moieties. A laser-induced proton pulse was used to characterize the proton-binding properties of bacterioopsin-containing membranes and of sarcoplasmic reticulum. Different protonation and deprotonation processes occurred. The liberation of protons from pyranine dye and the protonation of the membranes were independent of temperature; the reprotonation of pyranine and proton release from the membranes were temperature dependent. In the cases of membrane-free and membrane-containing systems, the activation enthalpies and entropies were calculated from the decay rates. The activation enthalpy of 16 kJ/mol for reprotonation of pyranine in membrane-free solution is characteristic for a diffusion-controlled process. The value for the membrane-containing systems was nearly double, suggesting that the buffering moieties of the membrane surfaces strongly bind the protons, raising the activation enthalpies. This is possibly an effect of the Coulomb cages formed from closely located proton acceptor sites. The activation entropies were positive in all cases.

Buffer effects on electric signals of light-excited bacteriorhodopsin mutants.

The effects of glycyl-glycine and bis-trispropane buffers on the light-excited electric signals due to proton motion in the molecule were studied for the bacteriorhodopsin (bR) mutants D38R, D96N, E204Q, R227Q, D85N, D85T, R82Q/D85N, and D85N/D96N in purple membranes and for delipidated purple membrane containing the wild-type bR. The results show additional charge motion caused by the buffers in all cases. Arrhenius parameters calculated from the temperature dependence of the difference signals (with buffer minus without buffer) are similar to the parameters found for the wild-type bR in the case of these buffers: the values of the activation enthalpies are mostly in the range 25-50 kJ/mol; all the activation entropies are negative. The results are evaluated with the cluster hypothesis outlined previously.

Frequency decoding of fast calcium oscillations by calpain.

We report the development of a novel procedure for generating fast, high-frequency Ca2+ oscillations in vitro and the frequency-dependent activation of m-calpain, the Ca2+-activated intracellular cysteine protease. The procedure is based upon liberating Ca2+ from a cage, DM-Nitrophen, by repetitive UV laser pulses and its concomitant binding by a 'slow' chelator, 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA). It is shown that a full control over the pattern of oscillations can be readily achieved because the half-life of individual spikes is determined by DOTA concentration and pH, whereas peak amplitude can be adjusted by light intensity. Frequency is only limited by the physical parameters of the light source. The sensitivity of calpain activation to the frequency of Ca2+ oscillations was monitored by the cleavage of microtubule-associated protein 2, a very sensitive physiological substrate of the enzyme. One hundred transients at a peak Ca2+ concentration of 10 microM were presented at various pH values and frequencies ranging from 1 to 50 Hz. At pH 6.0 and 7.0 significant activation occurred at high frequencies (20 and 50 Hz), but here Ca2+ accumulated due to the overlap of transients; at low frequencies (1 and 3 Hz) where Ca2+ accumulation was negligible, there was no calpain activation. At pH 8.0, where individual transients do not overlap even at 50 Hz, frequency-dependence of activation is seen when calpain is sensitized to Ca2+ by autolysis and by the addition of a phospholipid, phosphatidylinositol-4,5-bisphosphate. Our results show that calpain is sensitive to the frequency of fast Ca2+ oscillations in vitro, which is of potential physiological significance.

Electric signals of light excited bacteriorhodopsin mutant D96N.

The study of mutant D96N played an important role in understanding proton translocation by light driven bacteriorhodopsin. Our measurement of photoelectric current for single and double flash illumination revealed new details of the photocycle of this mutant. With double flash excitation we found an intermediate absorbing near the wavelength of the ground state of bacteriorhodopsin (bR) but pumping in the opposite direction. This intermediate has the same lifetime as the species described by Zimányi et al. [Proc. Natl. Acad. Sci. USA 96 (1999) 4414-4419] and was assigned to early recovery of a fraction of the ground state after excitation. Because the electric response does not reconcile with that of the ground state, we tentatively assign it to the L intermediate or to an intermediate similar in absorption to bR (bR').

Buffer effects on electric signals of light-excited bacteriorhodopsin.

Buffers change the electric signals of light-excited bacteriorhodopsin molecules in purple membrane if their concentration and the pH of the low-salt solution are properly selected. "Positive" buffers produce a positive component, and "negative" buffers a negative component in addition to the signals due to proton pumping. Measurement of the buffer effects in the presence of glycyl-glycine or bis-tris propane revealed an increase of approximately 2 and a change of sign and a decrease to approximately -0.5 in the translocated charge in these cases, respectively. These factors do not depend on temperature. The Arrhenius parameters established from the evaluation of the kinetics indicate activation enthalpies of 35-40 kJ/mol and negative activation entropies for the additional signals. These values agree with those found by surface-bound pH-sensitive probes in the search of the timing of proton release and uptake. The electric signals were also measured in the case of D(2)O solutions with similar results, except for the increased lifetimes. We offer a unified explanation for the data obtained with surface-bound probes and electric signals based on the clusters at extracellular and cytoplasmic sites of bacteriorhodopsin participating in proton release and uptake.

Photoelectric response of the N intermediate of bacteriorhodopsin and its mutant T46V.

Double flash experiments were performed in order to gain information about the characteristics of the N intermediates of the photocycle of bacteriorhodopsin. The N intermediates of wild-type bacteriorhodopsin and mutant T46V were excited at different delay times after the first laser flash which induced the photocycle and the electric responses were registered. These electric signals revealed that charge motions occurred in both cases, though charge translocation, i.e. H(+) pumping, could not be observed. The delay time dependence of the electric signals is characterized by two distinct processes corresponding to two substates of the N intermediates.

Interpretation of the spatial charge displacements in bacteriorhodopsin in terms of structural changes during the photocycle.

We have recently introduced a method, made possible by an improved orienting technique using a combination of electric and magnetic fields, that allows the three-dimensional detection of the intramolecular charge displacements during the photocycle of bacteriorhodopsin. This method generates electric asymmetry, a prerequisite for the detection of electric signal on the macroscopic sample, in all three spatial dimensions. Purple membrane fragments containing bacteriorhodopsin were oriented so that their permanent electric dipole moment vectors were perpendicular to the membrane plane and pointed in the same direction. The resulting cylindrical symmetry was broken by photoselection, i. e., by flash excitation with low intensity linearly polarized light. From the measured electric signals, the three-dimensional motion of the electric charge center in the bacteriorhodopsin molecules was calculated for the first 400 microseconds. Simultaneous absorption kinetic recording provided the time-dependent concentrations of the intermediates. Combining the two sets of data, we determined the discrete dipole moments of intermediates up to M. When compared with the results of current molecular dynamics calculations, the data provided a decisive experimental test for selecting the optimal theoretical model for the proton transport and should eventually lead to a full description of the mechanism of the bacteriorhodopsin proton pump.

Electrooptical measurements on purple membrane containing bacteriorhodopsin mutants.

Electrooptical measurements on purple membrane containing the wild-type and 10 different bacteriorhodopsin mutants have shown that the direction of the permanent electric dipole moment of all these membranes reverses at different pH values in the range 3.2-6.4. The induced dipole moment and the retinal angle exhibit an increased value at these pHs. The results demonstrate that the bacteriorhodopsin protein makes an important contribution to the electrooptical properties of the purple membrane.

Orientation of purple membrane in combined electric and magnetic fields.

The orientation of purple membrane in gels for photoelectric measurements is relatively poor, when they are prepared with the standard technique of applying a DC electric field and rapid polymerization. We have improved it by adding a high magnetic field (17.5 T) and increasing the viscosity of the membrane suspension. This process has resulted so far in a 3-fold increase of the photoelectric signals obtained. The magnetic susceptibility of purple membrane was determined.

Non-proton ion release in purple membrane.

Large conductivity changes have been measured during the photocycle of bacteriorhodopsin in purple membrane. These phenomena were explained as being due to the occurrence of large-scale non-proton ion release. Here we show that these conductivity changes do not appear if the purple membrane is immobilized. We propose an alternative hypothesis that explains the presence of conductivity change in suspensions and their absence in gels, as well as several related effects suggesting that the observed conductivity changes are due to alteration of the polarizability of purple membrane during the photocycle.

Alternative translocation of protons and halide ions by bacteriorhodopsin.

Bacteriorhodopsin (bR568) in purple membrane near pH 2 shifts its absorption maximum from 568 to 605 nm forming the blue protein bRacid605, which no longer transports protons and which shows no transient deprotonation of the Schiff base upon illumination. Continued acid titration with HCl or HBr but not H2SO4 restores the purple chromophore to yield bRHCl564 or bRHBr568. These acid purple forms also regain transmembrane charge transport, but no transient Schiff base deprotonation is observed. In contrast to bR568, no rate decrease of the bRacidpurple transport kinetics is detected in 2H2O; however, the transport rate decreases by a factor of approximately 2 in bRHBr568 compared with bRHCl564. The data indicate that in the acid purple form bR transports the halide anions instead of protons. We present a testable model for the transport mechanism, which should also be applicable to halorhodopsin.

Fast components of the absorption changes of bacteriorhodopsin at 275 nm and 296 nm.

A very fast component (life time 0.2 microsecond) was found in the flash-induced absorption changes of bacteriorhodopsin (bR) at 275 nm and 296 nm. This result was obtained by measuring the absorption changes at well defined delay times after the exciting laser flash (590 nm, 20 ns pulse duration). For this purpose a second laser flash was used as the monitoring beam. The very fast absorption changes of bR in the UV range are due to the rapid perturbation of the opsin moiety near the chromophore, as a result of the all-trans to 13-cis isomerization of the retinal taking place on the same time scale.

Angle of the retinal of bacteriorhodopsin in blue membrane.

The electric dichroism of purple and cation-depleted (blue) membrane was measured in a.c. electric fields at saturation. A decrease of 5.5 degrees in the direction of the chromophore transition moment with respect to the membrane normal was found upon removal of cations from purple membrane.

The bacteriorhodopsin proton pump: effect of crosslinkings of lysine residues.

All six available lysine residues in bacteriorhodopsin were amidinated with dimethyl-3,3'-dithiobispropionimidate, which is a crosslinking agent. The photocycle was studied by measuring light absorption and electric signals. The data show an essential change in the photocycle: instead of single components, the rise of the signal due to the M intermediate can be decomposed into two components, and the decay into three. The life-times and the intensities of these components and in general the proton pumping activity of bacteriorhodopsin depend only negligibly upon pH. Changes upon removing the crosslinks are not significantly different from those in the crosslinked samples. The lysine residues therefore may not be considered of primary importance in proton translocation.

Effect of cross linkers on the bacteriorhodopsin photocycle.

A general behavior of bacteriorhodopsin in purple membranes from Halobacterium halobium has been observed upon modification resulting in cross-linking of carboxyl and lysine groups. The rise of the M-intermediate contained two components with approximately 50-50% intensity; its decay showed three components with approximately 25-50-25% intensity respectively in a pH range of 5-9. The significance of these remarkably similar data with respect to the proton translocation mechanism in bacteriorhodopsin is that chemical modification allows us to conclude that disturbing parts of the hypothetical "proton conducting chain" does not inhibit proton translocation.