RESUMEN

Twenty-seven mutant bacteriorhodopsin's were screened to determine the PKa for reversal of the permanent electric dipole moment. The photoelectric response of an aqueous purple-membrane suspension was used to determine the direction of the purple-membrane dipole moment as a function of pH. The pK(a) for the dipole reversal of wild-type bacteriorhodopsin is 4.5. Six of the 27 mutant bacteriorhodopsin's were found to have a pK(a) for dipole reversal larger than that of wild-type bacteriorhodopsin. Two of these mutants, L93T and L93W, involve a neutral amino acid substitution in the interior of the protein. The direction of the purple-membrane permanent electric dipole moment is determined by the purple-membrane surface charge asymmetry. We conclude that these two substitutions, which do not involve charge replacement, alter the pK(a) for the reversal of the purple-membrane surface charge asymmetry. We suggest that these changes to the pK(a) are due to altered protein folding at the surface of the purple-membrane induced by single-site substitutions in the protein interior.

Asunto(s)

RESUMEN

Here we report the first observation of hyper-Rayleigh light scattering from bacteriorhodopsin in the form of an aqueous suspension of unoriented purple membranes. A typical purple membrane suspension used in our experiments contains approximately 10(8) randomly oriented purple membranes. Each purple membrane contains approximately 10(5) bacteriorhodopsin molecules in a two-dimensional crystallinearray. Hyper-Rayleigh light scattering is observed when the purple membrane suspension is illuminated with light that has a wavelength of 1064 nm. We propose that the 532-nm scattered light from each of the bacteriorhodopsin molecules in a single purple membrane is coherent, and that the scattered light from different purple membranes is incoherent. This proposal is supported by the following experimental observations: (a) the 532-nm light intensity is proportional to the square of the incident power, (b) the intensity of the 532-nm signal is linearly proportional to the concentration of purple membrane in solution, (c) the scattered 532-nm light is incoherent, (d) the scattered 532-nm light intensity decreases if the size of the purple membranes is reduced while the bacteriorhodopsin concentration is kept constant, and (e) the 532-nm light is due to the retinal chromophore of the bacteriorhodopsin molecule. The ratio of horizontal polarized hyper-Rayleigh scattered light to vertically polarized hyper-Rayleigh scattered light gives the angle (23 ± 4°) of the retinal axis with respect to the plane of the purple membrane. The hyperpolarizability of the bacteriorhodopsin molecule is found to be 5 ± 0.4 × 10(-27) esu.

RESUMEN

The initial photoinduced charge separation in bacteriorhodopsin is shown to occur in <5 ps. This result is obtained by measuring the photovoltage rise time in an oriented film of bacteriorhodopsin (BR). A dye laser syncronously pumped by an Argon ion cw mode locked laser is used to produce 3-ps light pulses which, after passing through a dye amplifier chain, photoexcite the BR sample. The photovoltage transient is detected by an ultra-fast Josephson junction digital sampling oscilloscope with liquid-helium-cooled input circuitry.

Asunto(s)

RESUMEN

The order of proton uptake and release in an aqueous suspension of purple membrane in response to a light flash has been investigated at lowered pH. pH indicator dyes and a flash spectrophotometer were used for the study. At pH 6.6 it was found that the release of protons from the purple membrane precedes uptake, as reported by other investigators. At pH 5.9, 4.9, and 4.1 it was also found that release precedes uptake. These results are not in agreement with those of previous investigators.

Asunto(s)

RESUMEN

The temperature dependence of the photovoltage developed by a model membrane containing bacteriorhodopsin (BR) is studied. The model membrane is formed by first coating a thin Teflon sheet with lipid and then fusing BR vesicles to it. The time course of the photoresponse is resolved down to 1 microsecond. The photoresponse is taken to be a sum of exponentials. Exponential time constants and amplitudes are determined by an analysis of the photoresponse with a photovoltage vs. log time plot, correlation filter, and nonlinear least-squares routine. The photovoltage is taken to be the sum of three exponentials but only two of the three time constants are resolved. Both are temperature dependent and indicate a thermally activated transport process. The corresponding activation energies are 55 kJ/mol and 62 kJ/mol. Since the photovoltage is proportional to charge times displacement the corresponding charge displacements are 11 and 34 A assuming a total displacement of 45 A. The remaining exponential term corresponds to a small negative transient in the photovoltage that has a rise time less than 1 microsecond even at -20 degrees C. The calculated charge displacement is estimated to be less than 2 A.

Asunto(s)

RESUMEN

The short-circuit photoresponse of a bacteriorhodopsin-based photoactive membrane is studied. The membrane is formed by first coating a Teflon membrane with lipid and then fusing bacteriorhodopsin vesicles to it. An incandescent light source was used to obtain the rise time of the photocurrent in response to a step-function illumination. A fast response, less than 1 ms, characterizes the initial rise and decay of the photocurrent. The trailing edge of the rise and trailing edge of the decay each exhibit different time constants both greater than 1 ms. These slower components show a sensitivity to membrane charging, the presence of diethylether in the bathing solution, and the presence of a charged cation complex in the lipid region. The photoresponse is not analyzed by means of the usual equivalent electrical circuit, but rather in terms of image charges in the conducting electrolyte bathing the membrane. Further experiments using a pulsed laser (pulse width less than 1 microseconds) resolve at least three time constants in the photoresponse: 0.057 ms, 1.06 ms, and 13 ms. Three distinct charge displacements (4.4, 7.5, and 33.1 A) are derived from the data, each corresponding to one of the above time constants.

Asunto(s)

RESUMEN

The short-circuit photoresponse of model membranes containing bacteriorhodopsin to short (35 ms) and long (3.5 s) light pulses is described. It is shown that if the light pulse is short compared with the charging and discharging times of the model membrane, the temporal response of the light-driven proton pump can be measured. Photoactive planar model membranes were formed both from biomolecular lipid membranes and from solid 6-micrometers thick Teflon septa coated with lipid and bacteriorhodopsin. The kinetic response of the pump is independent of the planar model membrane system in which it is incorporated. Experimental evidence indicates that the shape of the leading and trailing edges of the photoresponse curve for the pump deviates from simple exponential behavior. The short-circuit photoresponse of spinach chloroplast in a planar model membrane was also studied for comparison purposes.

Asunto(s)

RESUMEN

We have measured the light-induced short-circuit current generated by a planar membrane containing bacteriorhodopsin incorporated by vesicle fusion. The experimental results are consistent with an equivalent electrical circuit analogue that assumes that the vesicles remain intact after fusion and that the current generator equivalent of the light-driven proton pump is linearly dependent on bias voltage. The transient response to light of the planar membrane has also been examined. Slow response times are seen to be associated with the capacitive charging and discharging of the fused vesicles. A study of the leading edge of the light response curve of the planar membrane yields information about the transient response of the light-driven proton pump. We propose that the translocation of protons across the membrane is associated with a first-order process characterized by a rate constant lambda.

Asunto(s)

RESUMEN

The light-induced electrical current generated by black lipid membranes containing bacteriorhodopsin from Halobacterium halobium has been measured directly. It is shown that a measurement of membrane potential can also be used to obtain the proton pump current developed during illumination. Evidence is presented that the charge movement across the membrane is associated with the release of protons in the photoreaction cycle of bacteriorhodopsin. The time variation of the pump current when the light is turned on suggests the rapid depopulation of some initially occupied state.

Asunto(s)