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 <i>all</i>-<i>trans</i>, 15-<i>anti</i> and 13-<i>cis</i>, 15-<i>syn</i> 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

N-Terminal Truncation Does Not Affect the Location of a Conserved Tryptophan in the BLUF Domain of AppA from <i>Rhodobacter sphaeroides</i>

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

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

Raman Optical Activity Reveals Carotenoid Photoactivation Events in the Orange Carotenoid Protein in Solution

The orange carotenoid protein (OCP) plays an important role in photoprotection in cyanobacteria, which is achieved by the photoconversion from the orange dark state (OCP^O) to the red active state (OCP^R). Using Raman optical activity (ROA), we studied the conformations of the carotenoid chromophore in the active sites of OCP^O and OCP^R. This ROA measurement directly observed the chromophore conformation of native OCP in solution, and the measurement of OCP^R first demonstrated the ROA spectroscopy for the transient species. For OCP^O, the spectral features of ROA were mostly reproduced by the quantum chemical calculation based on the crystal structure of the OCP. Within the spatial resolution (∼2 Å), a slight modification of the polyene-chain distortion improved the agreement between the observed and calculated ROA spectra. While the crystal structure of OCP^R is not available, the ROA spectrum of OCP^R was reproduced by using the crystal structure of red carotenoid protein (RCP), an OCP^R proxy. The present results showed that the chromophore conformations in the crystal structures of OCP and RCP hold true for OCP^O and OCP^R in solution. Particularly, ROA spectroscopy of the native OCP^R provides a direct support for the 12 Å translocation of chromophore in the photoactivation, which was proposed by X-ray crystallography using RCP [R. L. Leverenz, M. Sutter, et al. <i>Science</i> <b>2015</b>, 348, 1463–1466]

Active Site Structure of Photoactive Yellow Protein with a Locked Chromophore Analogue Revealed by Near-Infrared Raman Optical Activity

Many biological cofactors, such as light-absorbing chromophores in photoreceptors, are intrinsically planar molecules. A protein environment, however, causes structural distortions of the cofactor, and such structural changes can lead to a modulation of chemical properties of the cofactor to maximize its biological activity. Here, we investigate the active site structure of photoactive yellow protein (PYP), a blue light photoreceptor that contains a <i>p</i>-coumaric acid (<i>p</i>CA) chromophore, by a near-infrared excited Raman optical activity (ROA). Specifically, we measured the ROA spectra of PYP, whose chromophore is replaced with a locked <i>p</i>CA analogue. Furthermore, we show that a spectral analysis based on quantum mechanical/molecular mechanical (QM/MM) calculations of the whole protein molecule is useful to obtain structural information from the observed ROA spectra. The use of the near-infrared ROA combined with QM/MM calculations is a novel and generally applicable spectroscopic tool to study the chromophore distortions within a protein environment

Spectroscopic Validation of Crystallographic Structures of a Protein Active Site by Chiroptical Spectroscopy

Out-of-plane distortions of a cofactor molecule in a protein active site are functionally important, and in photoreceptors, it has been proposed that they are crucial for spectral tuning and energy storage in photocycle intermediates. However, these subtle structural features are often beyond the grasp of structural biology. This issue is strikingly exemplified by photoactive yellow protein: its 14 independently determined crystal structures exhibit considerable differences in the dihedral angles defining the chromophore geometry, even though most of these are at excellent resolution. Here we developed a strategy to verify cofactor distortions in crystal structures by using quantum chemical calculations and chiroptical spectroscopy, particularly Raman optical activity and electronic circular dichroism spectroscopies. Based on this approach, we identify seven crystal structures with the chromophore geometries inconsistent with the experimentally observed data. The strategy implemented here promises to be widely applicable to uncovering cofactor distortions at active sites and to studies of reaction intermediates

Hydrogen Bonding Environment of the N3–H Group of Flavin Mononucleotide in the Light Oxygen Voltage Domains of Phototropins

The light oxygen voltage (LOV) domain is a flavin-binding blue-light receptor domain, originally found in a plant photoreceptor phototropin (phot). Recently, LOV domains have been used in optogenetics as the photosensory domain of fusion proteins. Therefore, it is important to understand how LOV domains exhibit light-induced structural changes for the kinase domain regulation, which enables the design of LOV-containing optogenetics tools with higher photoactivation efficiency. In this study, the hydrogen bonding environment of the N3–H group of flavin mononucleotide (FMN) of the LOV2 domain from <i>Adiantum</i> neochrome (neo) 1 was investigated by low-temperature Fourier transform infrared spectroscopy. Using specifically ¹⁵N-labeled FMN, [1,3-¹⁵N₂]­FMN, the N3–H stretch was identified at 2831 cm^–1 for the unphotolyzed state at 150 K, indicating that the N3–H group forms a fairly strong hydrogen bond. The N3–H stretch showed temperature dependence, with a shift to lower frequencies at ≤200 K and to higher frequencies at ≥250 K from the unphotolyzed to the intermediate states. Similar trends were observed in the LOV2 domains from <i>Arabidopsis</i> phot1 and phot2. By contrast, the N3–H stretch of the Q1029L mutant of neo1-LOV2 and neo1-LOV1 was not temperature dependent in the intermediate state. These results seemed correlated with our previous finding that the LOV2 domains show the structural changes in the β-sheet region and/or the adjacent Jα helix of LOV2 domain, but that such structural changes do not take place in the Q1029L mutant or neo1-LOV1 domain. The environment around the N3–H group was also investigated