Light-absorbing State Research Articles

Multiple Roles of a Conserved Glutamate Residue for Unique Biophysical Properties in a New Group of Microbial Rhodopsins Homologous to TAT Rhodopsin

TAT rhodopsin, a microbial rhodopsin found in the marine SAR11 bacterium HIMB114, uniquely possesses a Thr–Ala–Thr (TAT) motif in the third transmembrane helix. Because of a low pKa value of the retinal Schiff base (RSB), TAT rhodopsin exhibits both a visible light-absorbing state with the protonated RSB and a UV-absorbing state with the deprotonated RSB at a neutral pH. The UV-absorbing state, in contrast to the visible light-absorbing one, converts to a long-lived photointermediate upon light absorption, implying that TAT rhodopsin functions as a pH-dependent light sensor. Despite detailed biophysical characterization and mechanistic studies on the TAT rhodopsin, it has been unknown whether other proteins with similarly unusual features exist. Here, we identified several new rhodopsin genes homologous to the TAT rhodopsin of HIMB114 (TATHIMB) from metagenomic data. Based on the absorption spectra of expressed proteins from these genes with visible and UV peaks similar to that of TATHIMB, they were classified as Twin-peaked Rhodopsin (TwR) family. TwR genes form a gene cluster with a set of 13 ORFs conserved in subclade IIIa of SAR11 bacteria. A glutamic acid in the second transmembrane helix, Glu54, is conserved in all of the TwRs. We investigated E54Q mutants of two TwRs and revealed that Glu54 plays critical roles in regulating the RSB pKa, oligomer formation, and the efficient photoreaction of the UV-absorbing state. The discovery of novel TwRs enables us to study the universality and individuality of the characteristics revealed so far in the original TATHIMB and contributes to further studies on mechanisms of unique properties of TwRs.

English

Bacteriophytochromes (BphPs) are light-regulated biliprotein photoreceptors widely occurring in bacteria. BphPs exists in two spectral distinct states: the red-light absorbing state (Pr) and the far-red light-absorbing state (Pfr). BphPs are composed of an N-terminal photosensory core module and a light-controlled C-terminal histidine kinase module. The detailed mechanism on how photoconversion, initiated by a light-triggered isomerization of the biliverdin chromophore, is conducted from the N-terminal to the C-terminal domain and how the histidine kinase is modulated is still not clear. In this work Agp1 from Agrobacterium tumefaciens is used as a model phytochrome. The method of labeling full-length Agp1 mutants with single accessible Cys residue using fluorescence probes is described. Different absorption spectra showed that neither mutagenesis nor fluorescence labeling exerts significant effects on the photoconversion behavior of various Agp1 mutants. The histidine kinase activity assay revealed that the labeled holoproteins exhibit wild-types like autophosphorylation activity that depends on the Pr and the Pfr state. Fluorescence spectroscopic data suggested that fluorescence resonance energy transfer occurs from the donor Atto495 to the acceptor Atto565 in double-labeled Agp1-A362C, K514C, H554C, R603C, I653C, and V674C mutants. Red light irradiation gave rise to small changes on fluorescence emission intensity in some mutants but in some no remarkable inter-subunit distance changes occurred during the photo-conversion from Pr to Pfr in full-length Agp1. In contrast to double-labeled samples, significant changes in fluorescence intensity were observed in single-labeled samples.   Key words: Bacteriophytochromes (BphPs), C-terminal domain, photosensory, phytochrome. 

Phosphonofluoresceins: Synthesis, Spectroscopy, and Applications.

Xanthene fluorophores, like fluorescein, have been versatile molecules across diverse fields of chemistry and life sciences. Despite the ubiquity of 3-carboxy and 3-sulfonofluorescein for the last 150 years, to date, no reports of 3-phosphonofluorescein exist. Here, we report the synthesis, spectroscopic characterization, and applications of 3-phosphonofluoresceins. The absorption and emission of 3-phosphonofluoresceins remain relatively unaltered from the parent 3-carboxyfluorescein. 3-Phosphonofluoresceins show enhanced water solubility compared to 3-carboxyfluorescein and persist in an open, visible light-absorbing state even at low pH and in low dielectric media while 3-carboxyfluoresceins tend to lactonize. In contrast, the spirocyclization tendency of 3-phosphonofluoresceins can be modulated by esterification of the phosphonic acid. The bis-acetoxymethyl ester of 3-phosphonofluorescein readily enters living cells, showing excellent accumulation (>6x) and retention (>11x), resulting in a nearly 70-fold improvement in cellular brightness compared to 3-carboxyfluorescein. In a complementary fashion, the free acid form of 3-phosphonofluorescein does not cross cellular membranes, making it ideally suited for incorporation into a voltage-sensing scaffold. We develop a new synthetic route to functionalized 3-phosphonofluoresceins to enable the synthesis of phosphono-voltage sensitive fluorophores, or phosVF2.1.Cl. Phosphono-VF2.1.Cl shows excellent membrane localization, cellular brightness, and voltage sensitivity (26% ΔF/F per 100 mV), rivaling that of sulfono-based VF dyes. In summary, we develop the first synthesis of 3-phosphonofluoresceins, characterize the spectroscopic properties of this new class of xanthene dyes, and utilize these insights to show the utility of 3-phosphonofluoresceins in intracellular imaging and membrane potential sensing.

Dye-doped dual-frequency nematic cells as fast-switching polarization-independent shutters.

We present polarization-independent optical shutters with a sub-millisecond switching time. The approach utilizes dual-frequency nematics doped with a dichroic dye. Two nematic cells with orthogonal alignment are driven simultaneously by a low-frequency or high-frequency electric field to switch the shutter either into a transparent or a light-absorbing state. The switching speed is accelerated via special short pulses of high amplitude voltage. The approach can be used in a variety of electro-optical devices.

Conversion of phycocyanobilin-binding GAF domain to biliverdin-binding domain

Cyanobacteriochromes (CBCRs) are biliprotein photoreceptors that only exist in cyanobacteria and have a broad spectral response range from ultra-violet to far-red. The red/green-type CBCRs can show red/green reversible photoconversion via a covalently bound phycocyanobilin (PCB). In recent years, several CBCRs binding with not only PCB but also biliverdin (BV) have been discovered, which raises the possibility of CBCRs being applied as optogenetic tools. Through molecular modification, we hope to engineer BV-binding CBCRs responsive to the near-infrared spectral region (650–900 nm), of which the red/green type of CBCRs are suitable resources for experimentation. Here, we use Slr1393g3 (the third GAF domain of a red/green photoswitching CBCR from Synechocystis sp. PCC 6803) as a template to perform such molecular evolution using both random mutagenesis and site-directed mutagenesis. After several rounds of random mutagenesis, we obtained several BV-binding variants of Slr1393g3. These BV-binding variants have a maximal absorbance at ̃690 nm and a fluorescence at ̃720 nm. Additionally, some of them have remarkable photochromicity between a far-red light-absorbing state and a red light-absorbing state. Based on the primary amino acid sequence and structural models, the Phe474 surrounding ring D of BV is thought as a crucial site for chromophore selectivity.

Short-time dynamics and decay mechanism of 2(1H)-pyridinone upon excitation to the light-absorbing S4(21𝝅𝝅*) state

The excited-state structural dynamics and the decay mechanism of 2(1H)-pyridinone (NHP) after excitation to the S4(21ππ*) light-absorbing state were studied using resonance Raman spectroscopy and complete-active space self-consistent field (CASSCF) calculations. The B-band absorption cross-section and the corresponding absolute resonance Raman cross-sections were simulated using a simple model based on time-dependent wave-packet theory. The geometric structures of the singlet electronic excited states and their curve-crossing points were optimized at the CASSCF level of theory. The obtained short-time structural dynamics in easy-to-visualize internal coordinates were then compared with the CASSCF-predicted structural-parameter changes of S4(21ππ*)/S3(21nπ*)-MIN, S4(21ππ*)/S1(11nπ*)-MIN, and S4(21ππ*)-MIN. Our results indicate that the initial population of NHP in the S4 state bifurcates in or near the Franck-Condon region, leading to two predominant (S4S3-MIN and S4S1-MIN) internal conversion pathways. The lowest-lying S2(11ππ*) excited state is finally formed via subsequent internal conversions S3(21nπ*)/S2(11ππ*)-MIN and S1(11nπ*)/S2(11ππ*)-MIN. The enol-keto tautomeric mechanism does not seem to play a role. The decay mechanism in the singlet realm is proposed.

The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers

Agp1 is a canonical biliverdin-binding bacteriophytochrome from the soil bacterium Agrobacterium fabrum that acts as a light-regulated histidine kinase. Crystal structures of the photosensory core modules (PCMs) of homologous phytochromes have provided a consistent picture of the structural changes that these proteins undergo during photoconversion between the parent red light-absorbing state (Pr) and the far-red light-absorbing state (Pfr). These changes include secondary structure rearrangements in the so-called tongue of the phytochrome-specific (PHY) domain and structural rearrangements within the long α-helix that connects the cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) and the PHY domains. We present the crystal structures of the PCM of Agp1 at 2.70 Å resolution and of a surface-engineered mutant of this PCM at 1.85 Å resolution in the dark-adapted Pr states. Whereas in the mutant structure the dimer subunits are in anti-parallel orientation, the wild-type structure contains parallel subunits. The relative orientations between the PAS-GAF bidomain and the PHY domain are different in the two structures, due to movement involving two hinge regions in the GAF-PHY connecting α-helix and the tongue, indicating pronounced structural flexibility that may give rise to a dynamic Pr state. The resolution of the mutant structure enabled us to detect a sterically strained conformation of the chromophore at ring A that we attribute to the tight interaction with Pro-461 of the conserved PRXSF motif in the tongue. Based on this observation and on data from mutants where residues in the tongue region were replaced by alanine, we discuss the crucial roles of those residues in Pr-to-Pfr photoconversion.

Diversity of Active States in TMT Opsins.

Opn3/TMT opsins belong to one of the opsin groups with vertebrate visual and non-visual opsins, and are widely distributed in eyes, brains and other internal organs in various vertebrates and invertebrates. Vertebrate Opn3/TMT opsins are further classified into four groups on the basis of their amino acid identities. However, there is limited information about molecular properties of these groups, due to the difficulty in preparing the recombinant proteins. Here, we successfully expressed recombinant proteins of TMT1 and TMT2 opsins of medaka fish (Oryzias latipes) in cultured cells and characterized their molecular properties. Spectroscopic and biochemical studies demonstrated that TMT1 and TMT2 opsins functioned as blue light-sensitive Gi/Go-coupled receptors, but exhibited spectral properties and photo-convertibility of the active state different from each other. TMT1 opsin forms a visible light-absorbing active state containing all-trans-retinal, which can be photo-converted to 7-cis- and 9-cis-retinal states in addition to the original 11-cis-retinal state. In contrast, the active state of TMT2 opsin is a UV light-absorbing state having all-trans-retinal and does not photo-convert to any other state, including the original 11-cis-retinal state. Thus, TMT opsins are diversified so as to form a different type of active state, which may be responsible for their different functions.

X-ray Radiation Induces Deprotonation of the Bilin Chromophore in Crystalline D. radiodurans Phytochrome

We report that in the red light-absorbing (Pr) state, the bilin chromophore of the Deinococcus radiodurans proteobacterial phytochrome (DrBphP) is hypersensitive to X-ray photons used in typical synchrotron X-ray protein crystallography experiments. This causes the otherwise fully protonated chromophore to deprotonate without additional major structural changes. These results have major implications for our understanding of the structural and chemical characteristics of the resting and intermediate states of phytochromes and other photoreceptor proteins.

Crystallographic and Electron Microscopic Analyses of a Bacterial Phytochrome Reveal Local and Global Rearrangements during Photoconversion

Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photoreversible isomerization of their bilin chromophore to various signaling cascades. How changes in bilin conformation affect output by these photoreceptors remains poorly resolved and might include several species-specific routes. Here, we present detailed three-dimensional models of the photosensing module and a picture of an entire dimeric photoreceptor through structural analysis of the Deinococcus radiodurans phytochrome BphP assembled with biliverdin (BV). A 1.16-Å resolution crystal structure of the bilin-binding pocket in the dark-adapted red light-absorbing state illuminated the intricate network of bilin/protein/water interactions and confirmed the protonation and ZZZssa conformation of BV. Structural and spectroscopic comparisons with the photochemically compromised D207A mutant revealed that substitutions of Asp-207 allow inclusion of cyclic porphyrins in addition to BV. A crystal structure of the entire photosensing module showed a head-to-head, twisted dimeric arrangement with bowed helical spines and a hairpin protrusion connecting the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) and phytochrome-specific (PHY) domains. A key conserved hairpin feature is its anti-parallel, two β-strand stem, which we show by mutagenesis to be critical for BphP photochemistry. Comparisons of single particle electron microscopic images of the full-length BphP dimer in the red light-absorbing state and the photoactivated far-red light-absorbing state revealed a large scale reorientation of the PHY domain relative to the GAF domain, which alters the position of the downstream histidine kinase output module. Together, our data support a toggle model whereby bilin photoisomerization alters GAF/PHY domain interactions through conformational modification of the hairpin, which regulates signaling by impacting the relationship between sister output modules.

Fast polarisation-insensitive optical shutter supported by backflow in dichroic dye-doped dual-frequency liquid crystal

This paper describes a novel implementation of a dual-frequency liquid crystal optical shutter of the guest–host type. The transmissive state of the filter is obtained by applying a low-frequency electric field that brings the dichroic dye in a homeotropic orientation. The light-absorbing state is realised by a twisted planar configuration for which the absorption is quasi-independent of the polarisation. Switching between the two states occurs in about 1 ms and the devices show no scattering for wavelengths inside or outside the absorption band of the dichroic dye. Simulations and experiments reveal how a twisted state is obtained through the backflow phenomenon.

Domain Structure of a Unique Bacterial Red Light Photoreceptor as Revealed by Atomic Force Microscopy

ABSTRACTBacteriophytochromes (BphPs) are red-light photoreceptors found in photosynthetic and nonphotosynthetic bacteria that have been recently engineered as infrared fluorescent tissue markers. Light-induced, global structural changes are proposed to originate within their covalently bound biliverdin chromophore and propagate through the protein. Classical BphPs undergo reversible photoconversion between spectrally distinct light absorbing states, red (Pr) and far-red (Pfr), respectively. RpBph3 (P3), from Rhodopseudomonas palustris, photoconverts between a Pr and a unique near-red (Pnr) light-absorbing state. Due to size and photosensitivity of BphPs, structures of the intact proteins have not been resolved by nuclear magnetic resonance and/or X-ray crystallography. Therefore, structural details about the light and dark-adapted structures of the intact BphPs are not well understood at the molecular level. We have utilized fluid cell atomic force microscopy (AFM) to investigate the domain structure of intact P3 in its light-adapted state (Pnr). By varying the concentration of the protein, deposition time, and the ionic strength of the buffer, the aggregation of P3 on a mica surface can be controlled and single dimers may be observed in a biologically relevant media. Domain resolution has been achieved for several orientations of the dimer on the surface. The structural dimensions of the dimer have been compared to a modeled BphP in its intact form generated using PyMOL software. AFM experiments are currently underway to analyze the dark-adapted state (Pr) of P3 in order to observe the anticipated structural changes. Ultimately, the goal is to use AFM and other surface analytical methods such as scanning tunneling microscopy and electron microscopy to gain new insight into the unique photochemistry of P3.

Change in the energy of Jahn-Teller configurations of vacancy-donor complexes induced by uniaxial strain

Estimates are obtained for the energy splitting of equivalent Jahn-Teller configurations of the light-absorbing state of 〈vacancy Ga (V Ga)〉-〈tellurium (TeAs)〉 complexes in n-GaAs generated by uniaxial stress along the directions [111] and [001]. These estimates are based on measuring the stress dependence of the polarization of photoluminescence associated with these complexes at T⋍2 and 77 K. A phenomenological model of the complexes, which describes how the donor (TeAs) and the Jahn-Teller effect modify the initial t 2-orbitals of the vacancy component of the complex, is discussed as the effect of uniaxial strain. This model makes it possible to relate measured values of the energy splitting of equivalent configurations relative to the splitting of the original t 2-state of the vacancy arising from the presence of the donor and the Jahn-Teller effect. Comparison of calculations with experimental data shows that the contribution of the Jahn-Teller effect to the formation of the light-absorbing state of the complex V GaTeAs exceeds the contribution due to the donor effect, although the two effects are comparable.