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Photoactive Yellow Protein ( PYP), discovered almost 20 years ago in Ectothiorhodospira (Halorhodospira) halophila,¹ is a 4-hydroxycinnamic acid-containing protein that functions as a blue-light photoreceptor in a behavioral avoidance response in this organism. During the past 10 years, PYP has become a model system for studies in photochemistry and protein folding, to the extent that it has become competitive with the rhodopsins. This is because PYP is small and very water-soluble, forms crystals readily (diffracting to high resolution), and shows excellent chemical- and photo-stability. These overall characteristics have allowed the application of an array of physicochemical techniques to analyze the biological function of PYP, i.e., the translation of a change of the configuration of its 4-hydroxycinnamic acid chromophore into an altered conformation of the surrounding protein. This has led to detailed insight into this process, both temporally and spatially, with respect to the structure of the transient intermediates involved, although we are still quite far from being able to track the position of all atoms in space, upon light activation of the protein in the relevant time domain. Nevertheless, the data already obtained may function as a calibration set in future work, to extend the time span of molecular dynamics simulations of conformational transitions in proteins to the time scale relevant for catalytic turnover. Occasionally, the application of multiple biophysical techniques has led to (seemingly) conflicting results. In one example, this has revealed the fact that the light-induced conformational transitions in this photoreceptor protein can become restricted by the mesoscopic context, e.g., via a crystal lattice. Other inconsistencies, such as those regarding the radius of gyration of the protein, still remain to be explained. Below, we discuss the spatial and temporal details of the series of steps initiated in PYP by a short pulse of blue light, as revealed with this array of biophysical techniques, thereby highlighting contributions from our own group.

The complex photophysics of the wild-type green fluorescent protein (GFP), one of the most popular fluorescent probes in biology, has been extensively documented in literature. The excited-state dynamics of GFP was explained by means of a model implying excited-state proton transfer (ESPT) and three forms of the chromophore, a protonated A form absorbing at 400 nm and two deprotonated I and B forms absorbing at around 475 nm. We report here a systematic picosecond time-resolved fluorescence study of the enhanced green fluorescent protein (EGFP) variant, carrying the Ser65-Thr and Phe64-Leu mutations. By means of multiple excitation wavelength time-resolved experiments, we were able to distinguish between the fluorescence decay times of the deprotonated I* and B* states (3.4 and 2.7 ns). Spectrally, we found the I form being red shifted in comparison with the B form, both in absorption and in emission. Evidence for an excited-state reaction, namely, proton transfer, is also reported. An additional protonated species is proposed in the photophysical scheme in order to explain the excited-state dynamics of EGFP on the basis of our results as well as previous reported data. Two alternative models are presented, both of them applicable also to the data reported in relation with wild-type GFP.

The production of triplet states in basic solution of tetrakis(tetrasulfonated-phenyl)porphyrin (TPPS4) is accompanied by a contraction of (10 ± 3) mL/mol as determined by laser-induced optoacoustic spectroscopy (LIOAS) at different temperatures. The contraction is reverted as the triplet decays. The volume changes should mainly originate in changes in the solvation sphere due to the entire process of ground state to triplet transition (for the contraction) and back to the ground state (for the expansion). The presence of oxygen has no major effect on the contraction and the expansion observed. The present study shows that TPPS4 is not suitable as a calorimetric reference, contrary to our own hypothesis in Heihoff et al. Photochem. Photobiol. 1990, 51, 635−641. It was also observed that triplet state formation of methylene blue and rose bengal is accompanied by a structural volume change of −1.5 and 1 mL/mol, respectively.

The photochromic equilibria between the ground state and the first intermediate of the native photoreceptors phytochrome A (phyA) of oat and photoactive yellow protein (PYP) of Ectothiorhodospira halophila have been studied by laser-induced optoacoustic spectroscopy, employing photon densities sufficiently high to reach saturation and to establish the photoequilibria between the red-light-absorbing phyA form, Pr, and the first intermediate, I700, and between the PYP ground state, pG, and the first intermediate, pR. The parameters for the photoequilibria Pr &rlarr2; I700 and pG &rlarr2; pR were determined by the fluence saturation curves of the structural volume change, ΔVr, analyzed with model functions taking photoselection into account. The quantum yield of the photoreversion, ΦI700→Pr = 0.22 ± 0.12, proved to be ca. 1.4 times larger than the known quantum yield of the forward photoreaction (assuming ΦPr→I700 = ΦPr→Pfr). This suggests that the chromophore-binding protein domain structures of Pr and I700 are quite similar. In contrast, the photoreversion quantum yield in PYP for pR → pG is small (ΦpR→pG = 0.07) compared with the known value for the thermal forward process to the signaling state pB (ΦpG→pR ≥ ΦpG→pB = 0.35). This is tentatively attributed to a main conformational change associated with the pG → pR phototransformation. The results of this study emphasize the need of considering photoequilibria in photoreceptors when working with high-fluence laser pulses.

Optical trapping was combined with transmission microscopy (TM), confocal and nonconfocal fluorescence scanning microscopy (CFSM and FSM, respectively), and confocal and nonconfocal time-resolved fluorescence spectroscopy (CTRFS and TRFS, respectively) to study latex particles and block copolymer micelles. Dye-labeled latex particles of various size, in polymer composite films as well as optically trapped in solution, were studied with CFSM to characterize the limits of the setup. CFSM revealed that the resolution in the x- and y-directions was near the theoretical limit, i.e., 200−250 nm. CTRFS on the labeled latex particles revealed that the decay time of the label was not influenced by the polymer matrix nor the optical trap. Poly(tert-butylstyrene-block-sodium methacrylate) micelles (diameter approximately 30−40 nm) in deuterated aqueous solutions could be optically trapped, this region of high copolymer micelle concentration being referred to as a trapped cluster. In the transmission images, trapped clusters of 1.5−2 μm diameter were detected. Fluorescence images were obtained using perylene as a fluorophore that is specifically dissolved within the block copolymer micelles. The size of the trapped cluster, estimated from TM and FSM images, increases with increasing irradiation time and power, respectively. In the TM images, the trapped cluster appears as a dark spot (low transmission) with a bright (high transmission) corona-like ring around it. The appearance of the corona is explained as a light deflection phenomenon; i.e., the trapped cluster acts as lens due to a lateral refractive index gradient. When the corona is taken into account when the diameter of the trapped clusters is calculated, a very good agreement is found between TM and FSM. Long irradiation times lead to the formation of large trapped clusters, which are stable for about 10 s, with diameters of several hundred nanometers, while, for short irradiation times, the trapped cluster is smaller and disappears within a time less than 1 s. With CFSM it could be shown that the trapped particle has a spot size of approximately 1.7 μm in the region of the IR laser focus, while the diameter extends up to 5 μm without using the confocal imaging capability. The reason for this is that the conditions for optical trapping are fulfilled not only in but also above and below the focal region. Due to the high numerical aperture, a dumbbell-like shape of the trapped cluster results.