Hiroyuki Ohtani

PICOSECOND LASER PHOTOLYSIS OF SQUID RHODOPSIN AT ROOM AND LOW TEMPERATURES

Abstract. Squid rhodopsin extracted with 2% digitonin (pH 10.5 or 7.0) was excited with a 347 nm light pulse from a mode‐locked ruby laser at room temperature. Within 19 ps after the excitation, absorbance at 430 nm due to hypsorhodopsin increased and subsequently decreased with a decay time of 45 ± 10 ps. Absorbance at 550 nm due to bathorhodopsin increased with a rise time of 50 ± 10 ps. These results are the first observations of hypsorhodopsin at room temperature and clearly show that hypsorhodopsin is a precursor of bathorhodopsin which has been considered to be the earliest photoproduct in the photobleaching process of rhodopsin.

Squid hypsorhodopsin and bathorhodopsin by a picosecond laser photolysis.

The formation of bathorhodopsin (formerly prelumirhodopsin) was first observed by Yoshizawa and Kiti, [ 11 who irradiated cattle rhodopsin at liquid nitrogen temperature (77 K). Afterwards, the spectra of batho-intermediates in various animals were measured by low temperature spectrophotometry [2]. Therefore it is generally believed that bathorhodopsin is the earliest photoproduct in the photobleaching process of rhodopsin. Recently it was found that irradiation of cattle [2] or squid rhodopsin [3] at liquid helium temperature yielded two thermolabile photoproducts hypsorhodopsin @,a,: cattle 430 nm, squid 446 nm) and bathorhodopsin (A,,,: cattle 543 nm, squid 534 nm). Now the question arises: which is an earlier photoproduct of rhodopsin, hypsorhodopsin or bathorhodopsin? In order to elucidate this problem, we tried a picosecond laser flash photolysis of squid rhodopsin using a 347 nm light pulse from a mode locked ruby laser.

Mechanical perturbation-induced fluorescence change of green fluorescent protein

The force curve measurement mode of the atomic force microscope (AFM) is a powerful experimental technique in biotechnology. However, it is more effective if the spectroscopic properties of the biomolecule in the contact area can be simultaneously measured. Thus, we developed a confocal laser scanning microscope/AFM system. In this study, we simultaneously measured the fluorescence spectra of green fluorescent protein with the application of an external force in order to investigate the stability and dynamics of the β-barrel structure. Consequently, the fluorescence was quenched by applying both compression and extension forces and the quenching efficiencies differed in each case.

Time-resolved fluorometry of purple membrane of Halobacterium halobium O640 and an O-like red-shifted intermediate Q

The photocycle of the light‐adapted purple membrane was studied with a time‐resolved fluorometry apparatus: fluorescence of the sample suspension (>660 nm) was pumped with a 633‐nm cw laser and the temporal change induced by a 532‐nm pulsed laser was measured with a photon‐counting‐type transient recorder. The formation and the decay of the O640 intermediate were clearly observed in the pH region between 4.0 and 11.4. A photochemical cycle of N560 was apparently driven in alkaline suspension (pH > 9.3). An O‐like fluorescent intermediate Q appears and decays with time constants of <0.1 ms and 1.7 ± 0.2 ms, respectively.

Photoreaction of N560 intermediate in the photocycle of bacteriorhodopsin.

Sophisticated measurements were made on the nanosecond time‐resolved absorbance change of the purple membrane of Halobacterium halobium under cw background light irradiation (440–800 nm, 11–441 mW/cm2). A red‐shifted transient species R660 (KN, Q) was found in alkaline conditions (pH > 9.3). Background light intensity effect shows that (i) R660 is photochemically formed from N560 intermediate which is accumulated under background light irradiation because of the elongated lifetime in alkaline suspension, and that (ii) the slow decaying M412 is not photochemically formed from N560 but from bR.

Picosecond fluorescence spectroscopy of the purple membrane of Halobacterium halobium in alkaline suspension

Abstract The fluorescence lifetime of the light-adapted purple membrane of Halobacterium halobium in alkaline suspension was measured with a femtosecond-pulse laser-synchroscan streak camera system ( 568 ). We examined the effect of pH and excitation power on the amplitude of the slow component and concluded that its origin is attributable to the Q intermediate, which is a photoproduct of the N intermediate formed in the later stage of the photocycle of bR 568 . We found that Q is the same species as pseudo-bacteriorhodopsin (p-bR).

Ultrafast Photoreaction Dynamics of a Light-Driven Sodium-Ion-Pumping Retinal Protein from Krokinobacter eikastus Revealed by Femtosecond Time-Resolved Absorption Spectroscopy

We report the first femtosecond time-resolved absorption study on ultrafast photoreaction dynamics of a recently discovered retinal protein, KR2, which functions as a light-driven sodium-ion pump. The obtained data show that the excited-state absorption around 460 nm and the stimulated emission around 720 nm decay concomitantly with a time constant of 180 fs. This demonstrates that the deactivation of the S1 state of KR2, which involves isomerization of the retinal chromophore, takes place three times faster than that of bacteriorhodopsin. In accordance with this rapid electronic relaxation, the photoproduct band assignable to the J intermediate grows up at ∼620 nm, indicating that the J intermediate is directly formed with the S1 → S0 internal conversion. The photoproduct band subsequently exhibits a ∼30 nm blue shift with a 500 fs time constant, corresponding to the conversion to the K intermediate. On the basis of the femtosecond absorption data obtained, we discuss the mechanism for the rapid photoreaction of KR2 and its relevance to the unique function of the sodium-ion pump.

Picosecond fluorescence spectroscopy of purple membrane in Halobacterium halobium with a photon-counting streak camera

Abstract Fluorescence lifetimes and spectra of native and deionized purple membranes of Halobacterium halobium at 22°C were measured to be blue-shifted transient previously found by absorption spectroscopy is attributed to bacteriorhodopsin in the lowest excited-singlet state. Ultraweak fluorescence of the light-adapted purple membrane with 2.5 × 10 −4 quantum yield could be detected even though the excitation pulse energy at 570 nm was reduced to 0.88 pJ (72 μW average power).

Development of Confocal Laser Scanning Microscope/Atomic Force Microscope System for Force Curve Measurement

We developed a confocal laser scanning microscope (CLSM) / atomic force microscope (AFM) system by combining an inverted CLSM with an AFM for simultaneous spectroscopic measurement and force curve measurement in liquid. We observed resonance energy transfer from donor dyes attached to an AFM probe to acceptor dyes immobilized on the substrate surface. We simultaneously performed the spectroscopic measurement of contact area and force curve measurement and demonstrated the experimental technique and its validity with these experimental results.

Fluorescence spectra of bacteriorhodopsin and the intermediates O and Q at room temperature

An unequivocal answer is given to the question of why the reported fluorescence spectra of bacteriorhodopsin (bR568) have been different from one another. The inconsistency is shown to arise from the accumulation of the fluorescent intermediates O and Q (K N) by cw excitation light. Their fractions in the photo‐stationary states depend on the excitation power and the suspension pH. We report the intermediate‐free fluorescence spectrum of bR568 obtained with a weak excitation source (632.8 nm, 5.3 × 1015 − 1.9 × 1016 photons cm−2 · s−1) and a near‐IR sensitive intensified photodiode array system. The fluorescence maxima of the spectra, F(λ) and , are located at 755 ± 10 nm and 12700 ± 200 cm−1, respectively. The spectrum of O is identical to that of the deionized purple membrane bR605(F max = 750 ± 5 nm, f max = 13,000 ± 100 cm−1). Q (K N) exhibits a blue‐shifted spectrum more than that of bR568, (F max < 720 nm, f max < 13,400 cm−1).