R. Tóth-Boconádi

Bacteriorhodopsin as a possible chloride pump

Purple membranes oriented and immobilized in gel show charge transfer at pH 0.55 if the pH is set by HCl. Current appears as laser flash driven transient and also as continuous current by quasi‐continuous illumination. If the pH value 0.55 is set by H2SO4 continuous current is not observed. The results suggest that bacteriorhodopsin may pump chloride ions at low pH.

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.

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.

Counterions and the bacteriorhodopsin proton pump

Theoretical and new experimental arguments are given to explain the reversal of photoelectric signals from purple membranes oriented and immobilized in gel due to the presence of TEMED. The continuous current induced by continuous illumination demonstrates a photoelement‐like behaviour, the polarity of which is reversed by TEMED. The data render the counterion‐collapse mechanism highly questionable.

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.

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.

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.

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.

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.

Excitation of the M intermediates of bacteriorhodopsin.

Protein electric response signals (PERS) of the M intermediates of wild‐type bacteriorhodopsin (bR) were recorded. Contrary to earlier findings reporting on a single‐phase response upon excitation of the M intermediates, a kinetic analysis of the signals revealed the existence of three components, the fastest and the slowest ones of negative, while the middle one of positive sign with respect to the normal direction of proton pumping. Based on proton motion indicator experiments and molecular dipole calculations, the components were assigned to proton transfer steps and conformational changes driving the bR molecule back from the M to the ground state upon blue light excitation. The fastest, negative pump component was assigned to the proton transfer from D85 to the Schiff base. The subsequent positive component was attributed to rearrangements in the protein core (in the vicinity of the retinal molecule), triggered by the primary proton transfer process. The slowest component was established to reflect charge rearrangements associated with proton uptake by the protein from the bulk.