Structural and Vibrational Characterization of the Chromophore Binding Site of Bacterial Phytochrome Agp1

Agp1 is a prototypical bacterial phytochrome from Agrobacterium fabrum harboring a biliverdin cofactor which reversibly photoconverts between a red‐light‐absorbing (Pr) and a far‐red‐light‐absorbing (Pfr) states. The reaction mechanism involves the isomerization of the bilin‐chromophore followed by large structural changes of the protein matrix that are coupled to protonation dynamics at the chromophore binding site. Histidines His250 and His280 participate in this process. Although the three‐dimensional structure of Agp1 has been solved at high resolution, the precise position of hydrogen atoms and protonation pattern in the chromophore binding pocket has not been investigated yet. Here, we present protonated structure models of Agp1 in the Pr state involving appropriately placed hydrogen atoms that were generated by hybrid quantum mechanics/molecular mechanics‐ and electrostatic calculations and validated against experimental structural‐ and spectroscopic data. Although the effect of histidine protonation on the vibrational spectra is weak, our results favor charge neutral H250 and H280 both protonated at Nε. However, a neutral H250 with a proton at Nε and a cationic H280 may also be possible. Furthermore, the present QM/MM calculations of IR and Raman spectra of Agp1 containing isotope‐labeled BV provide a detailed vibrational assignment of the biliverdin modes in the fingerprint region.     

Modeling of phytochrome absorption spectra

Phytochromes constitute one of the six well‐characterized families of photosensory proteins in Nature. From the viewpoint of computational modeling, however, phytochromes have been the subject of much fewer studies than most other families of photosensory proteins, which is likely a consequence of relevant high‐resolution structural data becoming available only in recent years. In this work, hybrid quantum mechanics/molecular mechanics (QM/MM) methods are used to calculate UV‐vis absorption spectra of Deinococcus radiodurans bacteriophytochrome. We investigate how the choice of QM/MM methodology affects the resulting spectra and demonstrate that QM/MM methods can reproduce the experimental absorption maxima of both the Q and Soret bands with an accuracy of about 0.15 eV. Furthermore, we assess how the protein environment influences the intrinsic absorption of the bilin chromophore, with particular focus on the Q band underlying the primary photochemistry of phytochromes. © 2013 Wiley Periodicals, Inc.     

Beiträge zur Chemie der Pyrrolpigmente, 83. Mitt.: Zum Einfluß geladener Zentren auf die Absorptionsspektren von 1,19-Bilindionen

Summary Model systems consisting of 10-phenyl-bilin-1,19-diones substituted in 2- and 4-positions of the phenyl ring with carboxy or amino groups are synthesized by condensating appropriately substituted benzaldehydes with dipyrrinones. These systems provide a defined geometrical relation between bilin chromophore and functional group which can be charged by deprotonation or protonation. Spectrophotometrical titrations of these systems reveal that the negative charge exerts no influence on the absorption spectrum of the bilin. In the case of the positive charge problems arising from conformational and association equilibria prevent to draw such a conclusion.

     

Direct differentiation of A‐ring single attachment versus A‐ and D‐ring double attachment of phycoerythrobilin chromophores to phycobiliproteins using MALDI mass spectrometry

Bilin chromophore attachment to phycobiliproteins is an enzyme‐catalyzed post‐translational modification process. Bilin‐lyases attach a bilin chromophore to their cognate protein through a thioether bond between the chromophore and a cysteine moiety. Bilin chromophores are attached to their phycobiliproteins through the 3¹ carbon of the bilin. Double attachment may also occur, and in this case, carbons 3¹ and 18¹ of the bilin are both forming covalent linkages to cysteine moieties. There is a mass spectrometric limitation when examining tryptic peptides containing two (or more) cysteines if one seeks to ascertain whether chromopeptides are singly or doubly attached. The problem is that singly and doubly attached chromopeptides appear at the same m/z value; thus, up until the present, only NMR analysis has been successful at determining whether the chromophore is singly or doubly attached. We report in this work a new, fast and accurate method for discriminating singly from doubly attached chromophores using MALDI‐TOF mass spectrometry. This method was developed from mass spectral analysis of chromopeptides that had undergone in vitro or in vivo attachment of bilin chromophores to phycobiliproteins. Distinction is based on a characteristic neutral loss that appears in the MALDI‐TOF mass spectrum only when the bilin is singly attached. Copyright © 2013 John Wiley & Sons, Ltd.     

Time‐Resolved Energetics of Photoprocesses in Prokaryotic Phytochrome‐Related Photoreceptors

Time‐resolved photoacoustics (PA) is uniquely able to explore the energy landscape of photoactive proteins and concomitantly detects light‐induced volumetric changes (ΔV) accompanying the formation and decay of transient species in a time window between ca. 20 ns and 5 μs. Here, we report PA measurements on diverse photochromic bilin‐binding photoreceptors of prokaryotic origin: (1) the chromophore‐binding GAF3 domain of the red (R)/green (G) switching cyanobacteriochrome 1393 (Slr1393g3) from Synechocystis; (2) the red/far red (R/FR) Synechocystis Cph1 phytochrome; (3) full‐length and truncated constructs of Xanthomonas campestris bacteriophytochrome (XccBphP), absorbing up to the NIR spectral region. In almost all cases, photoisomerization results in a large fraction of energy dissipated as heat (up to 90%) on the sub‐ns scale, reflecting the low photoisomerization quantum yield (<0.2). This “prompt” step is accompanied by a positive ΔV₁ = 5–12.5 mL mol^?1. Formation of the first intermediate is the sole process accessible to PA, with the notable exception of Slr1393g3‐G for which ΔV₁ = +4.5 mL mol^?1 is followed by a time‐resolved, energy‐conserving contraction ΔV₂ = ?11.4 mL mol^?1, τ₂ = 180 ns at 2.4°C. This peculiarity is possibly due to a larger solvent occupancy of the chromophore cavity for Slr1393g3‐G.     

Prototropic tautomers of 5‐methylcytosine and enthalpy changes of their protonation,deprotonation, and deamination: Hybrid density functional B3LYP study

Molecular and thermodynamic properties such as geometric parameters, dipole moments, vibrational frequencies, the first ionization potentials, relative tautomerization energies, and tautomeric equilibrium constants of all prototropic tautomers of 5‐methylcytosine have been studied at the hybrid density functional level B3LYP/6‐31+G(d,p). The methylation on the C5 atom does not lead to significant geometric deformation of the pyrimidine structures of the corresponding tautomers of cytosine, which maintains the similar stability order. The tautomeric species 2‐oxo‐4‐amino [T(0)], 2‐hydroxy‐4‐amino [T(1‐2s) and T(1‐2t)], and trans‐2‐oxo‐4‐imino [T(3‐4t)] are predominated in the gas phase. The zwitterionic conformers of tautomerism [T(1‐4)] and protonation [P(4), P(1‐2s‐4), P(1‐2t‐4), and P(1‐3‐4)] are investigated for the first time due to their close relationship with deamination during genetic repair. Enthalpy changes (Δ_rH) of protonation, deprotonation, and deamination are calculated for these tautomeric species at room temperature; it is noted that the relative enthalpies [δ(ΔH)] of the tautomers are rationalized well in terms of a second‐order polynomial of the sum of the mean Δ_rH values of protonation and deprotonation processes. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002     

Effect of the PHY Domain on the Photoisomerization Step of the Forward Pr→Pfr Conversion of a Knotless Phytochrome

Phytochrome photoreceptors operate via photoisomerization of a bound bilin chromophore. Their typical architecture consists of GAF, PAS and PHY domains. Knotless phytochromes lack the PAS domain, while retaining photoconversion abilities, with some being able to photoconvert with just the GAF domain. Therefore, we investigated the ultrafast photoisomerization of the P_r state of a knotless phytochrome to reveal the effect of the PHY domain and its “tongue” region on the transduction of the light signal. We show that the PHY domain does not affect the initial conformational dynamics of the chromophore. However, it significantly accelerates the consecutively induced reorganizational dynamics of the protein, necessary for the progression of the photoisomerization. Consequently, the PHY domain keeps the bilin and its binding pocket in a more reactive conformation, which decreases the extent of protein reorganization required for the chromophore isomerization. Thereby, less energy is lost along nonproductive reaction pathways, resulting in increased efficiency.     

Tyrosine 263 in Cyanobacterial Phytochrome Cph1 Optimizes Photochemistry at the prelumi‐R→lumi‐R Step

We report a low‐temperature fluorescence spectroscopy study of the PAS‐GAF‐PHY sensory module of Cph1 phytochrome, its Y263F mutant (both with known 3D structures) as well as Y263H and Y263S to connect their photochemical parameters with intramolecular interactions. None of the holoproteins showed photochemical activity at low temperature, and the activation barriers for the Pr→lumi‐R photoreaction (2.5–3.1 kJ mol^?1) and fluorescence quantum yields (0.29–0.42) were similar. The effect of the mutations on Pr→Pfr photoconversion efficiency (Φ_Pr→Pfr) was observed primarily at the prelumi‐R S₀ bifurcation point corresponding to the conical intersection of the energy surfaces at which the molecule relaxes to form lumi‐R or Pr, lowering Φ_Pr→Pfr from 0.13 in the wild type to 0.05–0.07 in the mutants. We suggest that the E_a activation barrier in the Pr* S₁ excited state might correspond to the D‐ring (C19) carbonyl – H290 hydrogen bond or possibly to the hindrance caused by the C13¹/C17¹ methyl groups of the C and D rings. The critical role of the tyrosine hydroxyl group can be at the prelumi‐R bifurcation point to optimize the yield of the photoprocess and energy storage in the form of lumi‐R for subsequent rearrangement processes culminating in Pfr formation.     

Protonation of the chromophore in the photoactive yellow protein

The photoactive yellow protein (PYP) acts as a light sensor to its bacterial host: it responds to light by changing shape. After excitation by blue light, PYP undergoes several transformations, to partially unfold into its signaling state. One of the crucial steps in this photocycle is the protonation of p-coumaric acid after excitation and isomerization of this chromophore. Experimentalists still debate on the nature of the proton donor and on whether it donates the hydrogen directly or indirectly. To obtain better knowledge of the mechanism, we studied this proton transfer using Car-Parrinello molecular dynamics, classical molecular dynamics, and computer simulations combining these two methods (quantum mechanics/molecular mechanics, QMMM). The simulations reproduce the chromophore structure and hydrogen-bond network of the protein measured by X-ray crystallography and NMR. When the chromophore is protonated, it leaves the assumed proton donor, glutamic acid 46, with a negative charge in a hydrophobic environment. We show that the stabilization of this charge is a very important factor in the mechanism of protonation. Protonation frequently occurs in simplified ab initio simulations of the chromophore binding pocket in vacuum, where amino acids can easily hydrogen bond to Glu46. When the complete protein environment is incorporated in a QMMM simulation on the complete protein, no proton transfer is observed within 14 ps. The hydrogen-bond rearrangements in this time span are not sufficient to stabilize the new protonation state. Force field molecular dynamics simulations on a much longer time scale have shown which internal rearrangements of the protein are needed. Combining these simulations with more QMMM calculations enabled us to check the stability of protonation states and clarify the initial requirements for the proton transfer in PYP.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Common Structural Elements in the Chromophore Binding Pocket of the Pfr State of Bathy Phytochromes

Phytochromes are bimodal photoreceptors which, upon light absorption by the tetrapyrrole chromophore, can be converted between a red‐absorbing state (Pr) and far‐red‐absorbing state (Pfr). In bacterial phytochromes, either Pr or Pfr are the thermally stable states, thereby constituting the classes of prototypical and bathy phytochromes, respectively. In this work, we have employed vibrational spectroscopies to elucidate the origin of the thermal stability of the Pfr states in bathy phytochromes. Here, we present the first detailed spectroscopic analysis of RpBphP6 (Rhodopseudomas palustris), which together with results obtained for Agp2 (Agrobacterium tumefaciens) and PaBphP (Pseudomonas aeruginosa) allows identifying common structural properties of the Pfr state of bathy phytochromes, which are (1) a homogenous chromophore structure, (2) the protonated ring C propionic side chain of the chromophore and (3) a retarded H/D exchange at the ring D nitrogen. These properties are related to the unique strength of the hydrogen bonding interactions between the ring D N‐H group with the side chain of the conserved Asp194 (PaBphP numbering). As revealed by a comparative analysis of homology models and available crystal structures of Pfr states, these interactions are strengthened by an Arg residue (Arg453) only in bathy but not in prototypical phytochromes.     

Photo absorption of ‐coumaric acid in aqueous solution: RISM‐SCF‐SEDD theory approach

Photo absorption properties of p‐coumaric acid, the chromophore of photoactive yellow protein, in aqueous solution were investigated by means of reference interaction site model self‐consistent field with spatial electron density distribution (RISM‐SCF‐SEDD) method. RISM‐SCF‐SEDD is a combination methodology of electronic structure theory and statistical mechanics for molecular liquids. Here, time‐dependent density functional theory was coupled with RISM equation to study the electronic structure of p‐coumaric acid in aqueous system. Excitation energies of the chromophore in its neutral, two monoanionic and dianionic forms were computed to elucidate the effect of the deprotonation and solvation on the spectroscopic properties. We found that solvation strongly affects the excitation character of the chromophore, especially for phenolate anion and dianion. The free energy difference among the four protonation states is also discussed. © 2017 Wiley Periodicals, Inc.     

Tuning One‐Dimensional Nanostructures of Bola‐Like Peptide Amphiphiles by Varying the Hydrophilic Amino Acids

By combining experimental measurements and computer simulations, we here show that for the bola‐like peptide amphiphiles XI₄X, where X=K, R, and H, the hydrophilic amino acid substitutions have little effect on the β‐sheet hydrogen‐bonding between peptide backbones. Whereas all of the peptides self‐assemble into one dimensional (1D) nanostructures with completely different morphologies, that is, nanotubes and helical nanoribbons for KI₄K, flat and multilayered nanoribbons for HI₄H, and twisted and bilayered nanoribbons for RI₄R. These different 1D morphologies can be explained by the distinct stacking degrees and modes of the three peptide β‐sheets along the x‐direction (width) and the z‐direction (height), which microscopically originate from the hydrogen‐bonding ability of the sheets to solvent molecules and the pairing of hydrophilic amino acid side chains between β‐sheet monolayers through stacking interactions and hydrogen bonding. These different 1D nanostructures have distinct surface chemistry and functions, with great potential in various applications exploiting the respective properties of these hydrophilic amino acids.     

Complexation of a Macrocycle Containing Thiopyrimidine and Uracil Moieties with Dicarboxylic and Amino Acids in Various Organic and Aqueous Organic Solutions

The complexation of a macrocycle containing thiopyrimidine and uracil moieties (M) with amino acids and some dicarboxylic acids was studied by pH-metric, UV-VIS, ¹H NMR spectroscopy methods in chloroform, methanol, aqueous 1,4-dioxane, and biphasic water–chloroform media. The complexation of M with acids is too weak to solubilize them from the solid state into chloroform solutions containing M. The ¹H NMR spectra and pH-metric data of aqueous 1,4-dioxane (80 vol.%) reveal the pH-dependent 1:1 binding between M and the acids studied. The protonation of M is not a prerequisite for binding of fumaric, succinic, o-phtalic acids and the series of amino acids, whereas binding of maleic acid requires the protonation of both thiopyrimidine moieties of M. Therefore,M·(H⁺)₂ exhibits strong selectivity towards maleic acid in aqueous 1,4-dioxane and in biphasic water–chloroform media.     

Enhancement of Photophysical and Photosensitizing Properties of Flavin Adenine Dinucleotide by Mutagenesis of the C‐Terminal Extension of a Bacterial Flavodoxin Reductase

The role of the mobile C‐terminal extension present in Rhodobacter capsulatus ferredoxin–NADP(H) reductase (RcFPR) was evaluated using steady‐state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C‐terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild‐type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD‐binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge‐transfer quenching of ¹FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet‐ and triplet‐state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified.     

Highly Efficient Singlet–Singlet Energy Transfer in Light‐Harvesting [60,70]Fullerene–4‐Amino‐1,8‐naphthalimide Dyads

New C₆₀ and C₇₀ fullerene dyads formed with 4‐amino‐1,8‐naphthalimide chromophores have been prepared by the Bingel cyclopropanation reaction. The resulting monoadducts were investigated with respect to their fluorescence properties (quantum yields and lifetimes) to unravel the role of the charge‐transfer naphthalimide chromophore as a light‐absorbing antenna and excited‐singlet‐state sensitizer of fullerenes. The underlying intramolecular singlet–singlet energy transfer (EnT) process was fully characterized and found to proceed quantitatively (Φ_EnT≈1) for all dyads. Thus, these conjugates are of considerable interest for applications in which fullerene excited states have to be created and photonic energy loss should be minimized. In polar solvents (tetrahydrofuran and benzonitrile), fluorescence quenching of the fullerene by electron transfer from the ground‐state aminonaphthalimide was postulated as an additional path.     

Modified Cyclodextrins as Enantioselective Hosts for Amino Acids

Two modified β‐cyclodextrins, H‐2 and H‐3 , having a flexible appended moiety were studied for the chiral discrimination of the enantiomers of various amino acids by means of fluorescence as signaling option. These hosts quenched the fluorescence intensities of amino acids upon binding. The d‐ enantiomers were better recognized by these hosts. The association constants (K_s) and enantioselectivity factors (α) of the host?guest complexes were calculated.     

Tuning the Protein‐Induced Absorption Shifts of Retinal in Engineered Rhodopsin Mimics

Rational design of light‐capturing properties requires understanding the molecular and electronic structure of chromophores in their native chemical or biological environment. We employ here large‐scale quantum chemical calculations to study the light‐capturing properties of retinal in recently designed human cellular retinol binding protein II (hCRBPII) variants (Wang et al. Science, 2012 , 338, 1340–1343). Our calculations show that these proteins absorb across a large part of the visible spectrum by combined polarization and electrostatic effects. These effects stabilize the ground or excited state energy levels of the retinal by perturbing the Schiff‐base or β‐ionone moieties of the chromophore, which in turn modulates the amount of charge transfer within the molecule. Based on the predicted tuning principles, we design putative in silico mutations that further shift the absorption properties of retinal in hCRBPII towards the ultraviolet and infrared regions of the spectrum.     

Z and E isomers of butenedioic acid with 2‐amino‐1,3‐thiazole: 2‐amino‐1,3‐thiazolium hydrogen maleate and 2‐amino‐1,3‐thiazolium hydrogen fumarate

Maleic acid and fumaric acid, the Z and E isomers of butenedioic acid, form 1:1 adducts with 2‐amino‐1,3‐thiazole, namely 2‐amino‐1,3‐thiazolium hydrogen maleate (2ATHM), C₃H₅N₂S⁺·C₄H₃O₄⁻, and 2‐amino‐1,3‐thiazolium hydrogen fumarate (2ATHF), C₃H₅N₂S⁺·C₄H₃O₄⁻, respectively. In both compounds, protonation of the ring N atom of the 2‐amino‐1,3‐thiazole and deprotonation of one of the carboxyl groups are observed. The asymmetric unit of 2ATHF contains three independent ion pairs. The hydrogen maleate ion of 2ATHM shows a short intramolecular O—H...O hydrogen bond with an O...O distance of 2.4663 (19) Å. An extensive hydrogen‐bonded network is observed in both compounds, involving N—H...O and O—H...O hydrogen bonds. 2ATHM forms two‐dimensional sheets parallel to the ab plane, extending as independent parallel sheets along the c axis, whereas 2ATHF forms two‐dimensional zigzag layers parallel to the bc plane, extending as independent parallel layers along the a axis.