Is the photoactive yellow protein a UV-B/blue light photoreceptor?

UV light below 300 nm is shown to generate the first photocycle intermediate in the blue light photoreceptor Photoactive Yellow Protein. Fluorescence and ultrafast transient absorption measurements indicate two excitation pathways: UV-B absorption by the chromophore and Fluorescence Resonant Energy Transfer (FRET) from tryptophan and tyrosine residues.

Photodissociation pathways of gas-phase photoactive yellow protein chromophores.

The absorption dynamics of two model chromophores of the photoactive yellow protein were studied in gas-phase experiments. Using different time-resolving techniques with an overall sensitivity ranging from seconds down to a few nanoseconds, complex dynamics were revealed for the p -coumaric acid anion, involving both fragmentation and electron detachment as possible photoresponse channels. For the trans-thiophenyl-p-coumarate model, despite its more complex molecular structure, simpler decay dynamics showing only fragmentation were observed.

Spectral tuning of photoactive yellow protein.

We report a theoretical study on the optical properties of a small, water-soluble photosensory receptor, photoactive yellow protein (PYP). A hierarchical ab initio molecular orbital calculation accurately evaluated the optical absorption maximum of the wild-type, as well as the lambda(max) values of 12 mutants. Electronic excitation of the chromophore directly affects the electronic state of nearby atoms in the protein environment. This effect is explicitly considered in the present study. Furthermore, the spectral tuning mechanism of PYP was investigated at the atomic level. The static disorder of a protein molecule is intimately related to the complex nature of its energy landscape. By using molecular dynamics simulation and quantum mechanical structure optimization, we obtained multiple minimum energy conformations of PYP. The statistical distribution of electronic excitation energies of these minima was compared with the hole-burning experiment (Masciangioli, T. [2000] Photochem. Photobiol. 72, 639), a direct observation of the distribution of excitation energies.

Preparation of large crystals of photoactive yellow protein for neutron diffraction and high resolution crystal structure analysis.

The exact positions of all the hydrogen atoms in photoactive yellow protein (PYP) is important for understanding the molecular mechanism of the photoreaction because the protonation/deprotonation of certain amino acid residues and rearrangements in the hydrogen bond network are involved in the conformational changes of PYP. Neutron crystallography is one of the most effective methods to determine the hydrogen positions. However, a large crystal is required for neutron crystallography because a neutron-incident flux is quite limited. In addition, the crystal should be grown from heavy water to reduce the incoherent background from hydrogen. We prepared a large crystal of PYP (dimensions: 1.5 x 0.7 x 0.7 mm3) for neutron crystallography using ammonium sulfate with sodium chloride. The obtained large crystal gave X-ray diffraction spots up to 0.84 angstroms. Although some of the hydrogen atoms could be observed in the high resolution X-ray crystal structure, functionally important hydrogen atoms were impossible to see, indicating the importance of neutron crystallography. Thus, we optimized the crystallization conditions with heavy water and successfully obtained neutron diffraction spots up to 2.1 angstroms with the crystal in D2O.

Prokaryotic phototaxis.

Microorganisms have various mechanisms at their disposal to react to (changes in) their ambient light climate (i.e., intensity, color, direction, and degree of polarization). Of these, one of the best studied mechanisms is the process of phototaxis. This process can be described as a behavioral migration-response of an organism toward a change in illumination regime. In this chapter we discuss three of these migration responses, based on swimming, swarming, and twitching motility, respectively. Swimming motility has been studied using a wide range of techniques, usually microscopy based. We present a detailed description of the assays used to study phototaxis in liquid cultures of the phototrophic organisms Halobacterium salinarum, Halorhodospira halophila, and Rhodobacter sphaeroides and briefly describe the molecular basis of these responses. Swarming and twitching motility are processes taking place at the interface between a solid phase and a liquid or gas phase. Although assays to study these processes are relatively straightforward, they are accompanied by technical complications, which we describe. Furthermore, we discuss the molecular processes underlying these forms of motility in Rhodocista centenaria and Synechocystis PCC6803. Recently, it has become clear that also chemotrophic organisms contain photoreceptor proteins that allow them to respond to their ambient light climate. Surprisingly, light-modulated motility responses can also be observed in the chemotrophic organisms Escherichia coli and Acinetobacter calcoaceticus. In the light-modulated surface migration not only "che-like" signal transduction reactions may play a role, but in addition processes as modulation of gene expression and even intermediary metabolism.

The transient accumulation of the signaling state of photoactive yellow protein is controlled by the external pH.

The signaling state of the photoreceptor photoactive yellow protein is the long-lived intermediate I(2)'. The pH dependence of the equilibrium between the transient photocycle intermediates I(2) and I(2)' was investigated. The formation of I(2)' from I(2) is accompanied by a major conformational change. The kinetics and intermediates of the photocycle and of the photoreversal were measured by transient absorption spectroscopy from pH 4.6 to 8.4. Singular value decomposition (SVD) analysis of the data at pH 7 showed the presence of three spectrally distinguishable species: I(1), I(2), and I(2)'. Their spectra were determined using the extrapolated difference method. I(2) and I(2)' have electronic absorption spectra, with maxima at 370 +/- 5 and 350 +/- 5 nm, respectively. Formation of the signaling state is thus associated with a change in the environment of the protonated chromophore. The time courses of the I(1), I(2), and I(2)' intermediates were determined from the wavelength-dependent transient absorbance changes at each pH, assuming that their spectra are pH-independent. After the formation of I(2)' ( approximately 2 ms), these three intermediates are in equilibrium and decay together to the initial dark state. The equilibrium between I(2) and I(2)' is pH dependent with a pK(a) of 6.4 and with I(2)' the main species above this pK(a). Measurements of the pH dependence of the photoreversal kinetics with a second flash of 355 nm at a delay of 20 ms confirm this pK(a) value. I(2) and I(2)' are photoreversed with reversal times of approximately 55 micros and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pK(a) of approximately 6.1. Photoreversal from I(2)' dominates above the pK(a). The transient accumulation of I(2)', the active state of photoactive yellow protein, is thus controlled by the proton concentration. The rate constant k(3) for the recovery to the initial dark state also has a pK(a) of approximately 6.3. This equality of the equilibrium and kinetic pK(a) values is not accidental and suggests that k(3) is proportional to [I(2)'].

Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy.

Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates. Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP. In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger. The structural implications of these results are discussed.