D96N Bacteriorhodopsin Research Articles

2P242 In-situ光照射固体NMRによるバクテリオロドプシンD96N変異体の光中間体の捕捉とタンパク質構造変化の解明(18A.光生物:視覚・光受容,ポスター,日本生物物理学会年会第51回(2013年度))

2P242 In-situ光照射固体NMRによるバクテリオロドプシンD96N変異体の光中間体の捕捉とタンパク質構造変化の解明(18A.光生物:視覚・光受容,ポスター,日本生物物理学会年会第51回(2013年度))

Surface chemical functionalization of single walled carbon nanotubes with a bacteriorhodopsin mutant

In this work, single walled carbon nanotubes (SWNTs) have been chemically functionalized at their walls with a membrane protein, namely the mutated bacteriorhodopsin D96N, integrated in its native archaeal lipid membrane. The modification of the SWNT walls with the mutant has been carried out in different buffer solutions, at pH 5, 7.5 and 9, to investigate the anchoring process, the typical chemical and physical properties of the component materials being dependent on the pH. The SWNTs modified by interactions with bacteriorhodopsin membrane patches have been characterized by UV-vis steady state, Raman and attenuated total reflection Fourier transform infrared spectroscopy and by atomic force and transmission electron microscopy. The investigation shows that the membrane protein patches wrap the carbon walls by tight chemical interactions undergoing a conformational change; such chemical interactions increase the mechanical strength of the SWNTs and promote charge transfers which p-dope the nano-objects. The functionalization, as well as the SWNT doping, is favoured in acid and basic buffer conditions; such buffers make the nanotube walls more reactive, thus catalysing the anchoring of the membrane protein. The direct electron communication among the materials can be exploited for effectively interfacing the transport properties of carbon nanotubes with both molecular recognition capability and photoactivity of the cell membrane for sensing and photoconversion applications upon integration of the achieved hybrid materials in sensors or photovoltaic devices.

Peculiar Properties of Photoinduced Hydroxylaminolysis in Different Bacteriorhodopsin‐based Media Using O‐Substituted Hydroxylamines

The process of photoinduced hydroxylaminolysis has been re-examined in different bacteriorhodopsin (BR)-based media using O-substituted hydroxylamines, in particular, O-(4-nitrobenzyl) hydroxylamine hydrochloride (NBHA), O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (FBHA) and O-(t-butyl) hydroxylamine hydrochloride (BHA). Both wild type (WT) and D96N BR-based gelatine films and gels were studied. The expected increase in the bleaching rate of BR in gelatin films by using O-substituted hydroxylamines in place of HA was not achieved. On the other hand, it was shown that in gels HA derivatives NBHA and FBHA (as against HA itself) do provide about three- to four-fold higher bleaching rate. By contrast to that in films, D96N BR in gels demonstrates more effective bleaching as compared to WT BR. The plausible interpretation for the results is discussed in frames of reduced mobilities of large-sized molecules of O-substituted hydroxylamines in dehydrated media. FBHA- or NBHA-modified gels possess higher photosensitivity both with D96N and WT BR (as compared with that for HA-modified gels) and offer a potentiality for application as an irreversible-recording medium. As anticipated, it is specifically D96N BR gel modified with FBHA that may present a promising medium suitable for write-once recording thus extending the range of recording materials in the optical processing field.

Analysis of the recording efficiency of dynamic gratings in bacteriorhodopsin with the help of differential absorption spectra

The spectral distribution of the diffraction efficiency of amplitude-phase dynamic gratings in bacteriorhodopsin D96N is calculated based on the family of differential absorption spectra and the dispersion relations written in the difference form. The diffraction efficiencies at different wavelengths, and the diffraction efficiency ratio between the amplitude and phase components of a dynamic grating, are measured using the dynamic holography methods. It is shown that the experimental and calculation results agree well with each other within the absorption band of the initial trans state of bacteriorhodopsin.

Photoinduced Surface Potential Change of Bacteriorhodopsin Mutant D96N Measured by Scanning Surface Potential Microscopy

We report the direct measurement of photoinduced surface potential differences of wild-type (WT) and mutant D96N bacteriorhodopsin (BR) membranes at pH 7 and 10.5. Atomic force microscopy (AFM) and scanning surface potential microscopy (SSPM) were used to measure the BR membrane with the extracellular side facing up. We present AFM and SSPM images of WT and mutant D96N in which the light-dark transition occurred in the mid-scan of a single BR membrane. Photosteady-state populations of the M state were generated to facilitate measurement in each sample. The photoinduced surface potential of D96N is 63 mV (peak to valley) at pH 10.5 and is 48 mV at pH 7. The photoinduced surface potential of WT is 37 mV at pH 10.5 and approximately 0 at pH 7. Signal magnitudes are proportional to the amount of M produced at each pH. The results indicated that the surface potentials were generated by photoformation of surface charges on the extracellular side of the membrane. Higher surface potential correlated with a longer lifetime of the charges. A mechanistic basis for these signals is proposed, and it is concluded that they represent a steady-state measurement of the B2 photovoltage.

14‐Fluoro‐Bacteriorhodopsin Gelation Films for Dynamic Holography Recording¶

ABSTRACTThe first dynamic holography recording using 14‐fluoro‐(14‐F) bacteriorhodopsin (BR) gelatin films has been achieved. 14‐F BR is an artifical BR pigment made by reconstitution of bacterioopsin (native BR without chromophore) with synthetic 14‐F ratinal. Low‐intensity red light from a cw HE‐Ne laser was used for dynamic holography recording on the 14‐F wild type (WT) BR and 14‐F D96N mutant BR in gelatin films. There is not true comparing the diffraction efficiency for 14‐F D96N BR and 14‐F WT gelatin film, unlike the increase diffraction efficiency for D96N BR gelation film with native chromphore relative to the WT BR gelatin film with native chromophore. Pre‐illumination with blue light of the 14‐F BR gelatin films significantly increases the differaction effciency of both the the 14‐F WT and the 14‐F D96N BR pigments. The sequential application of blue and red laser beams indicates that 14‐F WT and the 14‐F D96N BR pigments. The sequential application of blue and red laser beams indicates taht 14‐F BR gelation films can be useful for optical memory.

14-Fluoro-Bacteriorhodopsin Gelatin Films for Dynamic Holography Recording¶

The first dynamic holography recording using 14-fluoro-(14-F) bacteriorhodopsin (BR) gelatin films has been achieved. 14-F BR is an artificial BR pigment made by reconstitution of bacterioopsin (native BR without chromophore) with synthetic 14-F retinal. Low-intensity red light from a cw He-Ne laser was used for dynamic holography recording on the 14-F wild type (WT) BR and 14-F D96N mutant BR in gelatin films. There is not true comparing the diffraction efficiency for 14-F D96N BR and 14-F WT gelatin film, unlike the increased diffraction efficiency for D96N BR gelatin film with native chromophore relative to the WT BR gelatin film with native chromophore. Pre-illumination with blue light of the 14-F BR gelatin films significantly increases the diffraction efficiency of both the 14-F WT and the 14-F D96N BR pigments. The sequential application of blue and red laser beams indicates that 14-F BR gelatin films can be useful for optical memory.

Photo-Electrochemistry of Ormosil Sandwiched D-96N Bacteriorhodopsin

The application of organically modified sol–gel glass (ORMOSIL) for stabilizing D96N bacteriorhodopsin is reported. The ormosil sandwiched bacteriorhodopsin shows high stability and better photoactivity than those made under similar conditions without ormosil. Three approaches for immobilizing Bacteriorhodopsin (BR) on either quartz plate or antimony tin oxide (ATO) electrode were investigated. System-1 was made by drying the BR preparation under ambient conditions. System-2 was made by drying the same amount of BR followed by sandwiching the dried BR suspension using a transparent ORMOSIL layer whereas system-3 was made by drying the BR suspension over a transparent layer of ormosil followed by the deposition of another layer of transparent ormosil over the dried BR, thus sandwiching BR between two layers of ORMOSILS. The transparent ormosil layer was made in aqueous acidic medium using 3-aminopropyltrimethoxysilane, phenyltrimethoxysilane and 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane. The characteristics of these three systems based on visual photography, UV-VIS spectroscopy (both in the presence and absence of external light) and photoelectrochemistry are reported. UV-VIS spectroscopy revealed excellent performance of BR when sandwiched between two layers of ORMOSILS (system-3) both in terms of photoactivity and stability for practical applications. The photo-electrochemistry of these three systems deposited on ATO electrodes is also reported. The photocurrent as a function of applied potential was recorded in the presence and absence of external light. The photocurrent showed increasing magnitude at negative potential and decreased from system-1 to system-3. The stability of ormosil sandwiched bacteriorhodopsin was found to exceed 1.5 years for practical applications.

Effect of Dehydration on Photoinduced Transformation in Gelatin Films Made With 14-Fluoro Bacteriorhodopsin Derivatives

Photoinduced transformation in gelatin films made with 14-fluoro bacteriorhodopsin derivatives, both wild-type (WT) and D96N mutant, were studied. Spectral and kinetic peculiarities for these two types of samples were compared over a wide range of relative humidity (9-92%). Analysis of the results considered two existing photoinduced processes that occur in suspensions and films of corresponding pigments. It was demonstrated that there is a range of humidity in which the performance of fluorine WT bacteriorhodopsin gelatin films may offer a technological advantage compared with fluorine D96N bacteriorhodopsin.

Light-induced changes in the absorption spectrum of bacteriorhodopsin under two-wavelength excitation

The results of spectrophotometric measurements of nonlinear light-induced changes in the absorption spectrum of bacteriorhodopsin D96N occurring upon simultaneous excitation at the wavelengths 633 and 441 nm in the excitation intensity range typical for recording of dynamic holograms are presented. The quantitative conditions under which the action of the radiation at one wavelength reduces the change in the optical density caused by the radiation at the other wavelength are determined.

Protein Flexibility and Conformational State: A Comparison of Collective Vibrational Modes of Wild-Type and D96N Bacteriorhodopsin

Far infrared (FIR) spectral measurements of wild-type (WT) and D96N mutant bacteriorhodopsin thin films have been carried out using terahertz time domain spectroscopy as a function of hydration, temperature, and conformational state. The results are compared to calculated spectra generated via normal mode analyses using CHARMM. We find that the FIR absorbance is slowly increasing with frequency and without strong narrow features over the range of 2–60 cm −1 and up to a resolution of 0.17 cm −1. The broad absorption shifts in frequency with decreasing temperature as expected with a strongly anharmonic potential and in agreement with neutron inelastic scattering results. Decreasing hydration shifts the absorption to higher frequencies, possibly resulting from decreased coupling mediated by the interior water molecules. Ground-state FIR absorbances have nearly identical frequency dependence, with the mutant having less optical density than the WT. In the M state, the FIR absorbance of the WT increases whereas there is no change for D96N. These results represent the first measurement of FIR absorbance change as a function of conformational state.

Recording of dynamic gratings in the nonlinear optical coating of a planar waveguide

The possibility of controlled energy exchange between interfering waveguide modes in a singlemode planar waveguide with a nonlinear optical coating is analyzed. As the coating, a suspension of bacteriorhodopsin D96N was used, which makes it possible to realize two spectrally separated mechanisms of recording and controlling dynamic gratings, i.e., the spatial modulation of the trans-cis excitation rate and the spatial modulation of the cis-trans relaxation rate. The method of phase-modulated beams was used to implement the energy exchange. The dynamic gratings in the coating were recorded by using both radiation with a wavelength within the absorption band of the trans state (630 nm) and radiation with a wavelength within the absorption band of the cis state (440 nm). Efficient control of the energy exchange between the waveguide modes by means of uniform exposure of their interference region to radiation with another wavelength was observed. A completely integral geometrical layout for optically controlled energy exchange was realized. The results obtained are compared with known data on energy exchange between beams in the bulk of a similar nonlinear medium.

Relationship between holographic and spectroscopic characteristics of photosensitive media with immobilized bacteriorhodopsin

The experimentally determined parameters of the photoresponse of a bacteriorhodopsin D96N suspension in glycerin are compared with the calculated values obtained from absorption spectra of this suspension. The kinetic parameters and saturation intensities are found to be different for the phase and amplitude components of the dynamic holographic grating in the medium under study. The effect of intermediate isomers of the photocycle of bacteriorhodopsin molecules is supposed to be responsible for the observed behavior of photoresponse. The photoresponse sensitivities and saturation parameters determined for either component of the grating from the energy transfer of the phase-modulated beams and from the spectral diagnostics of the medium are found to satisfactorily agree with each other.

Time-Resolved X-Ray Diffraction Reveals Movement of F Helix of D96N Bacteriorhodopsin during M-MN Transition at Neutral pH

D96N bacteriorhodopsin has two photointermediates with the deprotonated Schiff base: the M and MN intermediates. We measure the time-resolved x-ray diffraction of the D96N purple membrane after flash photoexcitation (pH 7.0, 25°C). The data clearly show the M-MN transition during the D96N photocycle. Low-resolution projection maps of these states show that the F helix of the MN intermediate shifts from its original position and this shift is much larger than that of the M intermediate. This indicates that the F helix moves in the M-MN transition of the D96N bacteriorhodopsin photocycle. Moreover, the existence of the MN intermediate in the D96N photocycle under neutral pH indicates that the MN intermediate is not peculiar to the alkaline condition. It is notable that the structural transition of M-MN is independent of the protonation state of the Schiff base. Therefore, the F helix movement precedes reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Our previous study showed that the M-MN transition is hydration-dependent and that the MN intermediate is more hydrated than the M intermediate. Considering this together with the present results, we conclude that the movement of the F helix causes hydration of the cytoplasmic side, which promotes the reprotonation of the Schiff base.

Holographic recording in a suspension of bacteriorhodopsin D96N with optically controllable response time

Get PDF Email Share Share with Facebook Tweet This Post on reddit Share with LinkedIn Add to CiteULike Add to Mendeley Add to BibSonomy Get Citation Copy Citation Text N. M. Kozhevnikov , A. E. Korolev, M. Yu. Lipovskaya, and V. N. Nazarov, "Holographic recording in a suspension of bacteriorhodopsin D96N with optically controllable response time," J. Opt. Technol. 67, 723- (2000) Export Citation BibTex Endnote (RIS) HTML Plain Text Citation alert Save article

Photoinduced anisotropy in chemically-modified films of bacteriorhodopsin and its genetic mutants

Photoinduced anisotropic properties of bacteriorhodopsin (BR) have attracted a lot of attention in the area of optical image processing. Here we report that chemical enhancement of wild-type BR, site-specific mutants D96E and D96N BR results in significant increase of the photoanisotropic response. The use of Asp-96 genetic mutants brings about just a slight increase in anisotropy and photosensitivity as compared to the effect of chemical modification of BR. To study the enhancement mechanism, the contributions of photoinduced birefringence and dichroism are estimated in the total photoanisotropic response. It is the increase of a total number of bleached BR molecules that are converted into a shortwavelength-absorbing intermediate M upon chemical modification of BR in films that results in the photoanisotropy increase. It is shown that photoinduced birefringence has positive extremum at around 635 nm and negative extremum – at around 460 nm. Maximum photoanisotropic response is detected with orthogonal polarization of the exciting beams of He–Ne and He–Cd lasers (B-M-type of photoinduced anisotropy).

Electrochemical studies on D96N bacteriorhodopsin and its application in the development of photosensors

The electrochemical studies on D96N bacteriorhodopsin (BR) are reported based on chronoamperometry using immobilized BR on antimony–tin oxide (ATO) electrode. The absorption spectra of immobilized BR on ATO electrode shows an absorption peak at 570 nm suggesting the stability of immobilized BR on ATO. Chronoamperometry of the immobilized BR on ATO is studied using a one-compartment homemade electrochemical cell having the arrangement of device for external light exposure upon immobilized BR on ATO as working electrode, Ag/AgCl as reference electrode and a Pt foil as auxiliary electrode. The chronoamperometry is studied in the presence and absence of light following two approaches by controlling the duration of light exposure to immobilized BR: (i) exposure to external yellow light for 1 s and (ii) exposure to external light for 12–20 s. The variation of photocurrent with time under these conditions are reported and discussed. The approach (i) has been used for the analysis of amine and ammonium compounds by measuring photocurrents in peak height mode. A good correlation between peak photocurrent and the concentration of amine and ammonium compounds is recorded. The data on approach (ii) results in forward photocurrent in the presence of light and backward photocurrent when the light is shut off. The effects of lysine, arginine and ammonium nitrate on variations of forward and backward photocurrents are reported. The dependence of photocurrent on applied potential vs. Ag/AgCl is reported. The calibration curves for the analysis of ammonium ion and amine compounds based on the measurement of photocurrent are reported.

Dependence of M, N and O states decay kinetics of D96N bacteriorhodopsin on amine and amino compounds and its application in chemical sensing

Abstract The dependence of the kinetics of M, N and O states decay of the D96N bacteriorhodopsin (BR) on (i) ammonium ion and (ii) an aromatic amine is reported. The kinetics of decay back to trans BR at 410, 530 and 640 nm, which are the absorption maximum for M, N and O states, respectively, in presence and absence of these analytes are studies for better understanding of the kinetics of M state decay back to trans BR as reported earlier [P.C. Pandey et al. Sensors and Actuators, 35–36 (1996) 470–474]. The half time (T1/2) of these decays and absorption spectra are measured at 410, 530 and 640 nm, respectively. The half time(T1/2) of these decay at 410 nm increases in the presence of amine whereas it decreases in the presence of amino compounds as compared with control. Similar trends have been observed at 530 and 640 nm. The reverse is observed on the measurement of absorption. The variation of these parameters are more apparent when measurements are carried out at 530 nm. The application of the kinetic data in the construction of chemical sensors is discussed and the analysis of ammonium ion and amine is reported.

Measurements of holographic properties of bacteriorhodopsin films

Several different bacteriorhodopsin (BR) films are characterized with respect to general holographic properties. Experimental measurements include diffraction efficiency and sensitivity as functions of the writing intensity and grating frequency, hologram thermal-decay behavior, diffraction efficiency as a function of the grating tilt within the film and the modulation depth, and estimates of the refractive-index change from the diffraction-efficiency data. The films studied include those made from wildtype BR and the genetic variants D96N and D96N/T46V. The film holographic properties were found to be relatively insensitive to the grating frequency and the grating-tilt angle. The diffraction efficiency dropped off more sharply as a function of the modulation depth than did a purely linear medium, and only the hydrated wildtype film exhibited significant behavior variation with different writing intensities because of its short M-state lifetime. The maximum diffraction efficiency measured was approximately 7.5% for a hydrated D96N BR film. We also find that the hydrated BR films exhibit significantly higher refractive-index modulation than do dry films.

Reversal in the kinetics of the M state decay of D96N bacteriorhodopsin: probing of enzyme catalyzed reactions

The kinetics of M state decay of the D96N bacteriorhodopsin (BR) in the presence of (i) ammonium ion and (ii) an aromatic amine is reported. The M state decay rate becomes faster in the presence of ammonium salt with a concomitant decrease in absorbance maxima at 410 nm. In the presence of an aromatic amine, a slower M state decay rate with an increase in absorbance maxima at 410 nm is observed. The variation in the kinetics of M-state decay is due to the change in the lifetime of the M-state associated with the conversion of M-intermediate to trans-BR. The decay rate of the M-state has also been found to be greatly affected in the presence of enzymatic reactions resulting in the formation of either an acid or a base. Three enzymatic reaction systems, urease, acetylcholinesterase and penicillinase, have been used to study the kinetics of M state of decay of D96N bacteriorhodopsin. The kinetic data are found to be dependent on the concentrations of the respective enzyme's substrate. Results of the studies using urea, acetylcholine and penicillin are reported. The enzymes were also immobilized together with D96N bacteriorhodopsin in sol-gel glass to develop a biosensor for the analysis of urea, acetylcholine and penicillin.