Variability in spectral absorption within cryptophyte phycobiliprotein types

Cryptophytes are known to vary widely in coloration among species. These differences in color arise primarily from the presence of phycobiliprotein accessory pigments. There are nine defined cryptophyte phycobiliprotein (Cr-PBP) types, named for their wavelength of maximal absorbance. Because Cr-PBP type has traditionally been regarded as a categorical trait, there is a paucity of information about how spectral absorption characteristics of Cr-PBPs vary among species. We investigated variability in primary and secondary peak absorbance wavelengths and full width at half max (FWHM) values of spectra of Cr-PBPs extracted from 75 cryptophyte strains (55 species) grown under full spectrum irradiance. We show that there may be substantial differences in spectral shapes within Cr-PBP types, with Cr-Phycoerythrin (Cr-PE) 545 showing the greatest variability with two, possibly three, subtypes, while Cr-PE 566 spectra were the least variable, with only ±1 nm of variance around the mean absorbance maximum of 565 nm. We provide additional criteria for classification in cases where the wavelength of maximum absorbance alone is not definitive. Variations in spectral characteristics among strains containing the same presumed Cr-PBP type may indicate differing chromophore composition and/or the presence of more than one Cr-PBP in a single cryptophyte species.     

Inter-domain crosstalk in the phytochrome molecules

Phytochromes are bifunctional photoreceptors with a two-domain structure, consisting of the N-terminal photosensory domain and the C-terminal regulatory domain. The photo-induced Pr <--> Pfr phototransformation accompanies subtle conformational changes, primarily triggered by the apoprotein-chromophore interactions in the N-terminal domain. The conformational signals are subsequently transmitted to the C-terminal domain through various inter-domain crosstalks, resulting in the interaction of the activated C-terminal domain with phytochrome interacting factors. Thus the inter-domain crosstalks play critical roles in the photoactivation of the phytochromes. Protein phosphorylation, such as that of Ser-598, is implicated in this process by inducing conformational changes and by modulating inter-domain signaling.     

Red fluorescent protein DsRed: Parametrization of its chromophore as an amino acid residue for computer modeling in the OPLS-AA force field

Topology of the neutral form of the DsRed fluorescent protein chromophore as a residue of [(4-cis)-2-[(1-cis)-4-amino-4-oxobutanimidoyl]-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl]acetic acid was calculated with OPLS-AA force field. Use of this topology and molecular dynamics simulation allows calculating the parameters of proteins that contain such residue in their polypeptide chains. The chromophore parameters were obtained by ab initio (RHF/6-31G**) quantum chemical calculations applying density functional theory (B3LYP). Using this chromophore, we have calculated the molecular dynamics trajectory of tetrameric fluorescent protein DsRed in solution at 300 K (4 nsec). Correctness of the chromophore parametrization was revealed by comparison of quantitative characteristics of the chromophore structure obtained from the molecular dynamic simulations of DsRed protein with the quantitative characteristics of the chromophore based on the crystallographic X-ray data of fluorescent protein DsRed (PDB ID: 1ZGO, 1G7K, and 1GGX), and also with the quantitative characteristics of the chromophore obtained by quantum chemical calculations. Inclusion of the neutral form of DsRed protein chromophore topology into the OPLS-AA force field yielded the extended force field OPLS-AA/DsRed. This force field can be used for molecular dynamics calculations of proteins containing the DsRed chromophore. The parameter set presented in this study can be applied for similar extension in any other force fields.     

Small-angle X-ray scattering reveals the solution structure of a bacteriophytochrome in the catalytically active Pr state

Phytochromes are light-sensing macromolecules that are part of a two component phosphorelay system controlling gene expression. Photoconversion between the Pr and Pfr forms facilitates autophosphorylation of a histidine in the dimerization domain (DHp). We report the low-resolution structure of a bacteriophytochrome (Bph) in the catalytic (CA) Pr form in solution determined by small-angle X-ray scattering (SAXS). Ab initio modeling reveals, for the first time, the domain organization in a typical bacteriophytochrome, comprising an chromophore binding and phytochrome (PHY) N terminal domain followed by a C terminal histidine kinase domain. Homologous high-resolution structures of the light-sensing chromophore binding domain (CBD) and the cytoplasmic part of a histidine kinase sensor allows us to model 75% of the structure with the remainder comprising the phytochrome domain which has no 3D representative in the structural database. The SAXS data reveal a dimeric Y shaped macromolecule and the relative positions of the chromophores (biliverdin), autophosphorylating histidine residues and the ATP molecules in the kinase domain. SAXS data were collected from a sample in the autophosphorylating Pr form and reveal alternate conformational states for the kinase domain that can be modeled in an open (no-catalytic) and closed (catalytic) state. This model suggests how light-induced signal transduction can stimulate autophosphorylation followed by phosphotransfer to a response regulator (RR) in the two-component system.     

Mapping the Structure of an Integral Membrane Protein under Semi-Denaturing Conditions by Laser-Induced Oxidative Labeling and Mass Spectrometry

Little is known about the structural properties of semi-denatured membrane proteins. The current study employs laser-induced oxidative labeling of methionine side chains in combination with electrospray mass spectrometry and optical spectroscopy for gaining insights into the conformation of bacteriorhodopsin (BR) under partially denaturing conditions. The native protein shows extensive oxidation at M32, M68, and M163, which are located in solvent-accessible loops. In contrast, M20 (helix A), M56/60 (helix B), M118 (helix D), M145 (helix E), and M209 (helix G) are strongly protected, consistent with the known protein structure. Exposure of the protein to acidic conditions leads to a labeling pattern very similar to that of the native state. The absence of large-scale conformational changes at low pH is in agreement with recent crystallography data. Solubilization of BR in SDS induces loss of the retinal chromophore concomitant with collapse of the binding pocket, thereby precluding solvent access to the protein interior. Tryptophan fluorescence data confirm the presence of a large protein core that remains protected from water. However, oxidative labeling indicates partial unfolding of helices A and D in SDS. Irreversible thermal denaturation of the protein at 100 °C induces a labeling pattern quite similar to that seen upon SDS exposure. Labeling experiments on refolded bacterioopsin reveal a native-like structure, but with partial unfolding of helix D. Our data suggest that noncovalent contacts with the retinal chromophore in native BR play an important role for the stability of this particular helix. Overall, the present work illustrates the viability of using laser-induced oxidative labeling as a novel tool for characterizing structural changes of membrane proteins in response to alterations of their solvent environment.     

Chromophores in photoproteins of a glowing squid and mollusk

Bioluminescence is a chemical reaction process for light emission in vivo. An organic substance is normally oxidized in the protein to obtain the energy required for the light emission. Determination of the structure of the substance is one of the most important parts of bioluminescent research. Photoproteins of a flying squid and a mollusk contain chromophores that are formed by connecting an apo-protein and dehydrocoelenterazine. The chromophore has a chemical structure that can emit light in a photoprotein. The structural analysis of the chromophores in the photoproteins is described.     

Evolution and the origin of the visual retinoid cycle in vertebrates

Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.     

CD exciton chirality method for determination of the absolute configuration of beta-hydroxy-alpha-amino acid derivatives

The absolute configuration of beta-hydroxy-alpha-amino acids was studied by CD exciton chirality method using 7-diethylaminocoumarin-3-carboxylate as a red-shifted chromophore. The CD spectra of bischromophoric derivatives of (S)-serine and (2S,3R)-threonine methyl esters (2 and 7) were compared with those of acyclic vic-aminoalcohols and diols (3--6 and 8--9). This study indicates that the polar carboxylate group of beta-hydroxy-alpha-amino acids makes them a unique subclass of vic-aminoalcohols. By combining the data of CD and NMR coupling constants, we are able to correlate their preferred conformer B and positive CD to the corresponding absolute configuration.