Masashi Unno

Structural refinement of a key tryptophan residue in the BLUF photoreceptor AppA by ultraviolet resonance Raman spectroscopy.

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. The BLUF domain is of special interest because it uses a rigid flavin rather than an isomerizable chromophore, such as a rhodopsin or phytochrome, for its light-activation process. Crystal and solution structures of several BLUF domains were recently obtained, and their overall structures are consistent. However, there is a key ambiguity regarding the position of a conserved tryptophan (Trp-104 in AppA), in that this residue was found either close to flavin (Trp(in) conformation) or exposed to the solvent (Trp(out) conformation). The location of Trp-104 is a crucial factor in understanding the photocycle mechanism of BLUF domains, because this residue has been shown to play an essential role in the activation of AppA. In this study, we demonstrated a Trp(in) conformation for the BLUF domain of AppA through direct observation of the vibrational spectrum of Trp-104 by ultraviolet resonance Raman spectroscopy, and also observed light-induced conformational and environmental changes in Trp-104. This study provides a structural basis for future investigations of the photocycle mechanism of BLUF proteins.

Exploring the active site structure of a photoreceptor protein by Raman optical activity.

We have developed a near-infrared excited Raman optical activity (ROA) spectrometer and report the first measurement of near-infrared ROA spectra of a light-driven proton pump, bacteriorhodopsin. Our results demonstrate that a near-infrared excitation enables us to measure the ROA spectra of the chromophore within a protein environment. Furthermore, the ROA spectra of the all-trans, 15-anti and 13-cis, 15-syn isomers differ significantly, indicating a high structural sensitivity of the ROA spectra. We therefore expect that future applications of the near-infrared ROA will allow the experimental elucidation of the active site structures in other proteins as well as reaction intermediates.

Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (phoborhodopsin) from Natronobacterium pharaonis.

Sensory rhodopsin II is a seven transmembrane helical retinal protein and functions as a photoreceptor protein in negative phototaxis of halophilic archaea. Sensory rhodopsin II from Natronomonas pharaonis (NpSRII) is stable under various conditions and can be expressed functionally in Escherichia coli cell membranes. Rhodopsins from microorganisms, known as microbial rhodopsins, exhibit a photocycle, and light irradiation of these molecules leads to a high-energy intermediate, which relaxes thermally to the original pigment after passing through several intermediates. For bacteriorhodopsin (BR), a light-driven proton pump, the photocycle is established as BR → K → L → M → N → O → BR. The photocycle of NpSRII is similar to that of BR except for N, i.e., M thermally decays into the O, and N has not been well characterized in the photocycle. Thus we here examined the second half of the photocycle in NpSRII, and in the present transient absorption study we found the formation of a new photointermediate whose absorption maximum is ∼500 nm. This intermediate becomes pronounced in the presence of azide, which accelerates the decay of M. Transient resonance Raman spectroscopy was further applied to demonstrate that this intermediate contains a 13-cis retinal protonated Schiff base. However, detailed analysis of the transient absorption data indicated that M-decay does not directly produce N but rather produces O that is in equilibrium with N. These observations allowed us to propose a structural model for a photocycle that involves N.

Vibrational assignment of the flavin-cysteinyl adduct in a signaling state of the LOV domain in FKF1.

LOV domains belong to the PAS domain superfamily, which are found in a variety of sensor proteins in organism ranging from archaea to eukaryotes, and they noncovalently bind a single flavin mononucleotide as a chromophore. We report the Raman spectra of the dark state of LOV domain in FKF1 from Arabidopsis thaliana. Spectra have been also measured for the signaling state, where a cysteinyl-flavin adduct is formed upon light irradiation. Most of the observed Raman bands are assigned on the basis of normal mode calculations using a density functional theory. We also discuss implication for the analysis of the infrared spectra of LOV domains. The comprehensive assignment provides a satisfactory framework for future investigations of the photocycle mechanism in LOV domains by vibrational spectroscopy.

Raman Optical Activity of a Cyclic Dipeptide Analyzed by Quantum Chemical Calculations Combined with Molecular Dynamics Simulations

Raman optical activity (ROA) measures the different intensity of right- and left-circularly polarized Raman scattered light and provides information on chirality associated with vibrational modes. Because of a high sensitivity to subtle structural and environmental changes, interpretations of ROA spectra usually rely on quantum chemical simulations. Recent advances in computational chemistry allow us to consider explicit solvent models that are derived from molecular dynamics (MD) simulations to compute the Raman and ROA spectra. An important concern for the explicit solvent models is the number of MD snapshots that lead to a good agreement between the observed and calculated spectra. In the present study, we measured the Raman and ROA spectra of cyclo(L-Ala-Gly) and then simulated the spectra using density functional theory combined with MD simulations. Although cyclo(L-Ala-Gly) is a relatively rigid cyclic molecule, boat-up and boat-down conformations were found from the MD calculations. Because the Raman spectra of the two conformations are similar except for a lower frequency region, ∼10 MD snapshots are capable of reproducing the main features of the observed Raman spectra. In contrast, a larger number of MD snapshots was required to reproduce the ROA spectra. In the middle freqency region of 800-1580 cm(-1), an average of ∼40 spectra led to good agreement between the observed and calculated spectra. On the other hand, the low (0-800 cm(-1)) and high (1580-1800 cm(-1)) frequency regions require more than 60 and 120 MD snapshots, respectively. The Raman and ROA spectra in the low frequency region are relatively broad, and such spectral features require a larger number of averaged spectra. The high frequency region of the spectra consists of an amide I band, which is primarily a C═O stretching vibration. Since both the ROA intensity and frequency of the amide I band are highly sensitive to structural and environmental differences, a large number of the spectra need to be averaged to reproduce the small negative features in the observed ROA spectra.

Raman Optical Activity Probing Structural Deformations of the 4-Hydroxycinnamyl Chromophore in Photoactive Yellow Protein.

Many biological cofactors, such as light-absorbing chromophores in photoreceptors, contain a π-electron system and are planar molecules. These cofactors are, however, usually nonplanar within a protein environment, and such structural distortions have been shown to be functionally important. Because the nonplanar structure makes the molecule chiral, Raman optical activity (ROA) provides a wealth of stereochemical information about the structural and conformational details of cofactors. The present study applied a near-infrared excited ROA to photoactive yellow protein, a blue light receptor. We successfully obtained the ROA spectra of the 4-hydroxycinnamyl chromophore embedded in a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.

Property and reactivity of fluoro(silyl)acetylenes and fluoro(stannyl)acetylenes.

Fluoro(silyl)acetylenes and fluoro(stannyl)acetylenes underwent a radical addition reaction of THF to furnish the corresponding fluorinated cyclic ethers in moderate to good yields. These intriguing addition reaction proved to proceed via a radical reaction mechanism.

Experimental Detection of the Intrinsic Difference in Raman Optical Activity of a Photoreceptor Protein under Preresonance and Resonance Conditions

Raman optical activity (ROA) is an advanced technique capable of detecting structural deformations of light-absorbing molecules embedded in chromophoric proteins. Resonance Raman (RR) spectroscopy is widely used to enhance the band intensities. However, theoretical work has predicted that under resonance conditions the ROA spectrum resembles the shape of the RR spectrum. Herein, we use photoactive yellow protein (PYP) to measure the first experimental data on the effect of changing the excitation wavelength on the ROA spectra of a protein. We observe a close similarity between the shape of the RR spectrum and the resonance ROA spectrum of PYP. Furthermore, we experimentally verify the theoretical prediction concerning the ratio of the amplitudes of the ROA and Raman spectra. Our data demonstrate that selecting an appropriate excitation wavelength is a key factor for extracting structural information on a protein active site using ROA spectroscopy.

N-terminal truncation does not affect the location of a conserved tryptophan in the BLUF domain of AppA from Rhodobacter sphaeroides.

The flavin-binding BLUF domains are a class of blue-light receptors, and AppA is a representative of this family. Although the crystal and solution structures of several BLUF domains have already been obtained, there is a key uncertainty regarding the position of a functionally important tryptophan (Trp104 in AppA). In the first crystal structure of an N-terminally truncated BLUF domain of AppA133 (residues 17-133), Trp104 was found in close proximity to flavin (Trp(in)), whereas in a subsequent structure with an intact N-terminus AppA126 (residues 1-126), Trp104 was exposed to the solvent (Trp(out)). A recent study compared spectroscopic properties of AppA126 and AppA133 and claimed that the Trp(in) conformation is an artifact of N-terminal truncation in AppA133. In this study, we compared the flavin vibrational spectra of AppA126 and AppA133 by using near-infrared excited Raman spectroscopy. In addition, the conformations as well as the environments of Trp104 were directly monitored by ultraviolet resonance Raman spectroscopy. These studies demonstrate that the N-terminal truncation does not induce the conformational switch between Trp(in) and Trp(out).

Transient Resonance Raman Spectroscopy of a Light-Driven Sodium-Ion-Pump Rhodopsin from Indibacter alkaliphilus

Sodium-ion-pump rhodopsin (NaR) is a microbial rhodopsin that transports Na+ during its photocycle. Here we explore the photocycle mechanism of NaR from Indibacter alkaliphilus with transient absorption and transient resonance Raman spectroscopy. The transient absorption data indicate that the photocycle of NaR is K (545 nm) → L (490 nm)/M (420 nm) → O1 (590 nm) → O2 (560 nm) → NaR, where the L and M are formed as equilibrium states. The presence of K, L, M, and O intermediates was confirmed by the resonance Raman spectra with 442 and 532 nm excitation. The main component of the transient resonance Raman spectra was due to L which contains a 13-cis retinal protonated Schiff base. The presence of an enhanced hydrogen out-of-plane band as well as its sensitivity to the H/D exchange indicate that the retinal chromophore is distorted near the Schiff base region in L. Moreover, the retinal Schiff base of the L state forms a hydrogen bond that is stronger than that of the dark state. These observations are consistent with a Na+ pumping mechanism that involves a proton transfer from the retinal Schiff base to a key aspartate residue (Asp116 in Krokinobacter eikastus rhodopsin 2) in the L/M states.