Masato Kumauchi

Identification of Six New Photoactive Yellow Proteins—Diversity and Structure–Function Relationships in a Bacterial Blue Light Photoreceptor†

Photoactive yellow proteins (PYP) are bacterial photoreceptors with a Per‐Arnt‐Sim (PAS) domain fold. We report the identification of six new PYPs, thus nearly doubling the size of this protein family. This extends the taxonomic diversity of PYP‐containing bacteria from photosynthetic to nonphotosynthetic bacteria, from aquatic to soil‐dwelling organisms, and from Proteobacteria to Salinibacter ruber from the phylum Bacteriodetes. The new PYPs greatly increase the sequence diversity of the PYP family, reducing the most prevalent pair‐wise identity from 45% to 25%. Sequence alignments and analysis indicate that all 14 PYPs share a common structure with 13 highly conserved residues that form the chromophore binding pocket. Nevertheless, the functional properties of the PYPs vary greatly—the absorbance maximum extends from 432 to 465 nm, the pKa of the chromophore varies from pH 2.8 to 10.2, and the lifetime of the presumed PYP signaling state ranges from 1 ms to 1 h. Thus, the PYP family offers an excellent opportunity to investigate how functional properties are tuned over a wide range, while maintaining the same overall protein structural fold. We discuss the implications of these results for structure–function relationships in the PYP family.

Locked chromophore analogs reveal that photoactive yellow protein regulates biofilm formation in the deep sea bacterium Idiomarina loihiensis

Idiomarina loihiensis is a heterotrophic deep sea bacterium with no known photobiology. We show that light suppresses biofilm formation in this organism. The genome of I. loihiensis encodes a single photoreceptor protein: a homologue of photoactive yellow protein (PYP), a blue light receptor with photochemistry based on trans to cis isomerization of its p-coumaric acid (pCA) chromophore. The addition of trans-locked pCA to I. loihiensis increases biofilm formation, whereas cis-locked pCA decreases it. This demonstrates that the PYP homologue regulates biofilm formation in I. loihiensis, revealing an unexpected functional versatility in the PYP family of photoreceptors. These results imply that I. loihiensis thrives not only in the deep sea but also near the water surface and provide an example of genome-based discovery of photophysiological responses. The use of locked pCA analogs is a novel and generally applicable pharmacochemical tool to study the in vivo role of PYPs irrespective of genetic accessibility. Heterologously produced PYP from I. loihiensis (Il PYP) absorbs maximally at 446 nm and has a pCA pK(a) of 3.4. Photoexcitation triggers the formation of a pB signaling state that decays with a time constant of 0.3 s. FTIR difference signals at 1726 and 1497 cm(-1) reveal that active-site proton transfer during the photocycle is conserved in Il PYP. It has been proposed that a correlation exists between the lifetime of a photoreceptor signaling state and the time scale of the biological response that it regulates. The data presented here provide an example of a protein with a rapid photocycle that regulates a slow biological response.

Chromophore Dynamics in the PYP Photocycle from Femtosecond Stimulated Raman Spectroscopy

Femtosecond stimulated Raman spectroscopy (FSRS) is used to examine the structural dynamics of the para-hydroxycinnamic acid (HCA) chromophore during the first 300 ps of the photoactive yellow protein (PYP) photocycle, as the system transitions from its vertically excited state to the early ground state cis intermediate, I0. A downshift in both the C7═C8 and C1═O stretches upon photoexcitation reveals that the chromophore has shifted to an increasingly quinonic form in the excited state, indicating a charge shift from the phenolate moiety toward the C9═O carbonyl, which continues to increase for 170 fs. In addition, there is a downshift in the C9═O carbonyl out-of-plane vibration on an 800 fs time scale as PYP transitions from its excited state to I0, indicating that weakening of the hydrogen bond with Cys69 and out-of-plane rotation of the C9═O carbonyl are key steps leading to photoproduct formation. HOOP intensity increases on a 3 ps time scale during the formation of I0, signifying distortion about the C7═C8 bond. Once on the I0 surface, the C7═C8 and C1═O stretches blue shift, indicating recovery of charge to the phenolate, while persistent intensity in the HOOP and carbonyl out-of-plane modes reveal HCA to be a cissoid structure with significant distortion about the C7═C8 bond and of C9═O out of the molecular plane.

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

The robustness of proteins against point mutations implies that only a small subset of residues determines functional properties. We test this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PER-ARNT-SIM (PAS) domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. We report high-throughput biophysical measurements on a complete Ala scan set of purified PYP mutants. The dataset of 1,193 values on active site properties, functional kinetics, stability, and production level reveals that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties. Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. PAS domain activation often involves conformational changes in α-helices linked to the PAS core. However, the mechanism of transmission and kinetic regulation of allosteric structural changes from the PAS domain to these helices is not clear. The Ala scan data reveal interactions governing allosteric switching in PYP. The photocycle kinetics is significantly altered by substitutions at 58 positions and spans a 3,000-fold range. Nine residues that dock the N-terminal α-helices of PYP to its PAS core regulate signaling kinetics. Ile39 and Asn43 are identified as part of a mechanism for regulating allosteric switching that is conserved among PAS domains. These results show that PYP combines robustness with a high degree of evolvability and imply production level as an important factor in protein evolution.

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.

Photoactive Yellow Protein Terahertz Response: Hydration, Heating and Intermediate States

Photoactive yellow protein (PYP) is a model system for studies on functional protein dynamics and the role of protein flexibility during function. Functional conformational change is initiated by anharmonic collective vibrational modes that absorb in the far infrared (FIR) or terahertz (THz) region. We have used THz time-domain spectroscopy (THz-TDS) to investigate changes in the flexibility of PYP with functional state change (initial pG state to pB photo intermediate) induced by dehydration and photo excitation for both thin films and solutions. We find that the THz absorbance follows the dehydration induced capture of the pB state, indicating that the loss of photo cycling with the pB dehydration transition may be associated with the loss of picosecond flexibility. For hydrated films we find that previous reports of THz sensitivity to the pG to pB transition are likely in error and either arise from system drift or heating effects. We find no change in the dielectric response with photo induced occupation of the pB state. We compare these results with computational results and find that the THz dielectric response is dominated by relaxational motions of the solvent and surface side chains.

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.

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.

Modulating Native-like Residual Structure in the Fully Denatured State of Photoactive Yellow Protein Affects Its Refolding

Residual structure in the fully unfolded state is a key element for understanding protein folding. We show that the residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid (pCA) chromophore. The exposure of total surface area and hydrophobic surface area upon unfolding was quantified by denaturant m values and heat capacity changes (ΔCp), respectively. The exposure of the buried surface area upon the unfolding of the acid-denatured state of PYP containing trans-pCA is ∼20% smaller than that of the native state. In contrast, for the partially unfolded pB photocycle intermediate containing cis-pCA, unfolding-induced exposure of the surface area is not decreased. These results show that pCA photoisomerization reduces residual structure in the fully unfolded state. Thus, residual structure in the fully unfolded state of PYP is under direct experimental control by photoexcitation. The sensitivity of the unfolded state to pCA isomerization provides a novel criterion that residual structure in the unfolded state of PYP is native-like, involving native-like protein-chromophore interactions. A largely untested prediction is that native-like residual structure facilitates the conformational search during folding. In the case of PYP, refolding from the less disordered fully unfolded state containing trans-pCA indeed is substantially accelerated. The burial of hydrophobic surface area in the fully unfolded state suggests that a significant part of the hydrophobic collapse process already has occurred in the denatured state.

Side-chain specific isotopic labeling of proteins for infrared structural biology: The case of ring-D4-tyrosine isotope labeling of photoactive yellow protein

An important bottleneck in the use of infrared spectroscopy as a powerful tool for obtaining detailed information on protein structure is the assignment of vibrational modes to specific amino acid residues. Side-chain specific isotopic labeling is a general approach towards obtaining such assignments. We report a method for high yield isotope editing of the bacterial blue light sensor photoactive yellow protein (PYP) containing ring-D(4)-Tyr. PYP was heterologously overproduced in Escherichia coli in minimal media containing ring-D(4)-Tyr in the presence of glyphosate, which inhibits endogenous biosynthesis of aromatic amino acids (Phe, Trp, and Tyr). Mass spectrometry of the intact protein and of tryptic peptides unambiguously demonstrated highly specific labeling of all five Tyr residues in PYP with 98% incorporation and undetectable isotopic scrambling. FTIR spectroscopy of the protein reveals a characteristic Tyr ring vibrational mode at 1515 cm(-1) that is shifted to 1436 cm(-1), consistent with that from ab initio calculations. PYP is a model system for protein structural dynamics and for receptor activation in biological signaling. The results described here open the way to the analysis of PYP using isotope-edited FTIR spectroscopy with side-chain specific labeling.