Kinko Tsuji

Structural changes in bacteriorhodopsin induced by electric impulses

Abstract Electric impulses of high field intensity (2 × 105 to 3 × 106 Vm−1, 1 to 20 μs duration) cause transient changes in the optical absorbance of suspended purple membranes of Halobacterium halobium. The electric dichroism at 1 m m NaCL, pH ≈ 6 and at 293K is dependent on field strength, pulse duration and wavelength of the monitoring, plane-polarized light in the range 400 to 650 nm. The optically detected processes are, however, independent of bacteriorhodopsin concentration, of ionic strenght and of the intensity of the monitoring light. These data together with the analysis of time course ands steady state of the reduced dichroism, suggest electric field-sensitive, intramemembraneous structural changes which lead to restricted orientation changes of the chromophore. A thoretical analysis of restricted orientation is developed and applied to the electro-optic data. As a result, it is found that the electric dichroism of purple membrane is associated with a large polarizability anisotropy of 2.4 × 10−30 Fm2 (2.2 × 10−14 cm3); the electric permanent dipole moment which is involved amounts to 4.7 × 10−28 Cm(140 Debye). The kinetic data suggest a cyclic reaction scheme with at least five different conformations. The high polarizability is probably due to displaceable ionic groups within the cooperative lattice of bacteriorhodopsin molecules in purple membranes.

Conformational flexibility of membrane proteins in electric fields. I. Ultraviolet absorbance and light scattering of bacteriorhodopsin in purple membranes.

Bacteriorhodopsin of halobacterial purple membranes exhibits conformational flexibility in high electric field pulses (1-30 x 10(5) V m(-1), 1-100 micros). High-field electric dichroism data of purple membrane suspensions indicate two kinetically different structural transitions within the protein; involving a rapid (approximately 1 micros) concerted change in the orientation of both retinal and tyrosine and/or tryptophan side chains concomitant with alterations in the local protein environment of these chromophores. as well as slower changes (approximately 100 micros) of the microenvironment of aromatic amino acid residues concomitant with pK changes in at least two types of proton-binding sites. Light scattering data are consistent with the maintenance of the random distribution of the membrane discs within the short duration of the applied electric fields. The kinetics of the electro-optic signals and the steep dependence of the relaxation amplitudes on the electric field strength suggest a saturable induced-dipole mechanism and a rather large reaction dipole moment of 1.1 x 10(-25) C m ( = 3.3 x 10(4) debye) per cooperative unit at E = 1.3 x 10(5) V m(-1), which is indicative of appreciable cooperativity in the probably unidirectional transversal displacement of ionic groups on the surfaces of and within the bacteriorhodopsin proteins of the membrane lattice. The electro-optic data of bacteriorhodopsin are suggestive of a possibly general, induced-dipole mechanism for electric field-dependent structural changes in membrane transport proteins such as the gating proteins in excitable membranes or the ATP synthetases.

Electric-field induced pK-changes in bacteriorhodopsin.

Bacteriorhodopsin, the only protein of the purple membranes of halobacteria [ 11, acts as a light-driven proton pump, translocating protons from the inner to the outer side of the plasma membrane [2]. The lightinduced asymmetric proton distribution contributes to the membrane potential by up to lo-40 mV [3]. Assuming a membrane thickness of 50 A, this potential difference corresponds to an electric field strength of 2-8 X lo6 V/m. Since the protein is polyionic it is possible that photocycle and proton transport are affected by a direct action of the membrane electric field on the conformation of bacteriorhodopsin. An electric field (l-30 X 10’ V/m and l-30 ps duration) causes a cycle of structural changes within the bacteriorhodopsin of purple membrane suspensions [4]. The data suggest an induced-dipole mechanism which involves a restricted rotational displacement of the chromophore by an angle of >2O”C toward the membrane normal [4]. We show here that the field-induced structural changes in purple membrane involve alterations of the pK-values of at least two types of proton binding sites of bacteriorhodopsin. The comparison of the time course of the pH changes indicates that the electric field-induced pK-shifts are opposite in direction to the light-induced pK-changes. This observation suggests a possible negative feedback of the increased electric membrane field on the proton transporting conformations of bacteriorhodopsin.

Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding

Magnesium binding to cation#x2010;depleted blue bacteriorhodopsin (b‐bR) was studied spectrophotometrically as well as by following stopped‐flow kinetics. There exist three kinetically different steps in the binding process, yielding purple bacteriorhodopsin (p‐bR). Since only the firtst step is dependent on the concentration of the reactants, the reaction scheme can be proposed as the simplest model, with MgbR being the first intermediate and ΣI denoting a set of successive intermediates. According to this model k 1, k −1 and k 2 are calculated to be 2.8 × 104 M−1 · s−1, 5.0 × 10 s−1 and 1 × 10−2 s−1, respectively.

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field-induced absorption changes of bacteriorhodopsin were studied with different samples of purple membranes which were prepared as randomly oriented and electrically oriented films of purple as well as cation-depleted blue bacteriorhodopsin. The absorption changes were proportional to the square of the field strength up to ≈300 kV/cm. The electric field from the intracellular side to the extracellular side of the purple bacteriorhodopsin induces a spectrum change, resulting in a spectrum similar to that of the cation-depleted blue bacteriorhodopsin. When the field was removed, the purple state was regenerated. The blue state was mainly affected by an electric field in the opposite direction, suggesting a reversible interaction with the Schiffs base bond of the retinal. Since the field-induced reaction of bacteriorhodopsin was observed in the presence of a concomitant steady ion flux, it is assumed that the generation of a local diffusion potential may play an important role in these spectral reactions. Although the fragments were fixed in the dried film, electric dichroism was observed. The dichroic contribution of the total absorbance change was about 15%. The angular displacement of the retinal transition moment was calculated to be 1.5° toward the membrane normal.

Electrooptical analysis of blue and of cation-regenerated bacteriorhodopsin

Blue bacteriorhodopsin was prepared by electrodialysis, cation-exchange chromatography and acidification. The electrooptical properties of these preparations compared to those of the native purple bacteriorhodopsin suggest that the blue bacteriorhodopsin has a smaller induced dipole moment than the native purple bacteriorhodopsin and that bound cations in the native bacteriorhodopsin stabilize the protein conformation in the membrane.Purple bacteriorhodopsin was regenerated by addition of potassium, magnesium or ferric ions to blue bacteriorhodopsin. Both spectrscopically and electrooptically the potassium- and ferric-regenerated samples are different from the native purple state. Although the magnesium-regenerated sample is spectroscopically similar to the native purple bacteriorhodopsin, the electrooptical properties are rather similar to those of the cation-depleted blue sample, suggesting that it is very difficult to re-stabilize protein structures once cations are depleted.

728—The ion-pair concept in macromolecular dynanmics

Abstract Iron-pairing appears to be a functionally essential interaction element in macromolecular structures such as protein, nucleic acids and membranes. Recent progress in understanding bioelectric field effects in macromolecular and cellular organizations in summarized in terms of the ion-paired concept. A rigorous general treatment of electric field effects in dipolar chemical systems is outlined in terms of a dielectrochemical potential.

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.

Electrooptical studies on proton-binding and -release of bacteriorhodopsin at various pH values

Abstract Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring of the absorbance change of pH indicators. There are, at least, two proton binding sites; one (site 1) releases protons and the other (site 2) takes up protons in the presence of the electric field. The pH dependence of the number of protons released from site 1 and taken up by site 2 indicates that the pK value of site 1 is shifted from 5.0 to 4.6 and that of site 2 from 4.5 to 5.5 by the externally applied electric field of 20 kV/cm. The rate constants of proton binding for site 1 and site 2 in the pH range from 4.7 to 7.3 are estimated to be 2 × 108M−1s−1 and 2 × 107M−1 s−1 respectively.

Interaction of Electric Fields with Membrane-Bound Polyionic Proteins

Recent progress in electro-optic instrumentation has led to experimental results which give new insight into the dynamic behavior of membrane-bound polyionic macromolecules, such as bacteriorhodopsin in purple membranes. Electric impulses of high field intensity (2×105 to 3×106 Vm−1,1 to 20 ps duration) cause transient changes in the optical absorbance of suspended purple membranes of Halobacterium halobium. The electric dichroism at 1 mM NaC1 pH ≅ 6 and at 293 K is dependent on field strength, pulse duration and wavelength of the monitoring, plane-polarized light in the range 400 nm to 650 nm. The optically indicated processes are, however, independent of bacteriorhodopsin concentration, of ionic strength and of the intensity of the monitoring light. These data and the analysis of time course and steady state of the reduced dichroism suggest electric field sensitive, intramembraneous structural changes which involve restricted orientation changes of the chromophore. A theoretical analysis of restricted orientation is developed and applied to the electro-optic data. As a result it is found that the electric dichroism of purple membranes is associated with a large induced dipole moment up to 7×10−26 Cm (2.1×104 Debye) which develops in a cooperative manner; the electric permanent dipole moment which is involved amounts to 4.7×10−28 Cm (140 Debye).