Conformation Of Chromophore Research Articles

Chromophore distortions in the bacteriorhodopsin photocycle: evolution of the H-C14-C15-H dihedral angle measured by solid-state NMR.

In recent years, structural information about bacteriorhodopsin has grown substantially with the publication of several crystal structures. However, precise measurements of the chromophore conformation in the various photocycle states are still lacking. This information is critical because twists about the chromophore backbone chain can influence the Schiff base nitrogen position, orientation, and proton affinity. Here, we focus on the C14-C15 bond, using solid-state nuclear magnetic resonance spectroscopy to measure the H-C14-C15-H dihedral angle. In the resting state (bR(568)), we obtain an angle of 164 +/- 4 degrees, indicating a 16 degrees distortion from a planar all-trans chromophore. The dihedral angle is found to decrease to 147 +/- 10 degrees in the early M intermediate (M(o)) and to 150 +/- 4 degrees in the late M intermediate (M(n)). These results demonstrate changes in the chromophore conformation undetected by recent X-ray diffraction studies.

On the bioactive conformation of the rhodopsin chromophore: absolute sense of twist around the 6-s-cis bond.

Incubation of opsin with synthetic 6-s-locked retinoids 2a and 2b only led to pigment formation from the alpha-locked 2a, the CD spectrum of which was similar to that of native rhodopsin (Rh). This establishes that the 6-s-bond of the chromophore in rhodopsin is cis, and that its helicity is negative. Earlier cross-linking studies showed that the 11-cis to all-trans photoisomerization occurring in the batho-Rh to lumi-Rh conversion induces a flip over of the side carrying the ring moiety. The GTP-binding assay of pigment Rh-(2a), incorporating retinal analogue 2a, has shown that its activity is 80% that of the native pigment. That is, the overall conformation around the 6-s bond is retained in the steps leading to G-protein activation.

Rebeccamycin analogues from Indolo[2,3-c]carbazole

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important series of antitumor drugs. In the course of structure–activity relationship studies, we report the synthesis of two new derivatives containing an indolo[2,3-c]carbazole chromophore instead of the conventional indolo[2,3-a]carbazole unit found in the natural metabolites. The N-methylated compound 8 containing one glucose residue behaves as a typical DNA intercalating agent, as judged from circular and electric linear dichroism measurements with purified DNA. In contrast, the bis-glycosylated derivative 7 containing a glucose residue on each indole nitrogen has lost its capacity to form stable complexes with DNA. DNA relaxation experiments reveal that the two drugs 7 and 8 have weak effects on human DNA topoisomerase I. The modified conformation of the indolocarbazole chromophore is detrimental to the stabilization of topoisomerase I–DNA complexes. The lack of potent topoisomerase I inhibition leads to decreased cytotoxicity but, however, we observed that the DNA-intercalating mono-glycosyl derivative 8 is about 5 times more cytotoxic than the bis-glycosyl analogue 7. The study suggests that the naturally-occurring indolo[2,3-a]carbazole skeleton should be preserved to maintain the topoisomerase I inhibitory and cytotoxic activities.

Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein.

To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.

Synthesis and solvatochromism of soluble polyethers containing isolated emissivep-aryl vinylene derivatives

Two soluble polyethers (η red > 0.4 dL/g) consisting of isolated emissive p-aryl vinylene derivatives have been synthesized and characterized. The introductions of ether linkages and aliphatic chains result in enhanced solubility in common organic solvents such as tetrahydrofuran (THF) and chloroform. The polyethers exhibit good thermal stability with onset decomposition temperatures at around 400 °C in nitrogen. The photoluminescence spectra of the two polyethers show a maximum peaks at 446 and 394 nm, respectively. The shifts of the photoluminescence maxima are controlled by the steric conformation of the emissive chromophores. On the other part, an interesting solvatochromism of three polyethers was observed in solution. The absorption maxima of dilute polymeric solutions exhibit bathochromism at first, and then hypsochromism with increasing solvent polarity. This may be due to the alteration of the electronic structure in these emissive polyethers.

Enantioselective binding of an 11-cis-locked cyclopropyl retinal. The conformation of retinal in bovine rhodopsin.

[formula: see text] The conformation of the retinal chromophore in rhodopsin is central for understanding the visual transduction process. The absolute twist around the 12-s bond of the chromophore in rhodopsin has been determined by studies with 11-cis-locked 11,12-cyclopropylretinal analogues (11S,12R)-2 and (11R,12S)-3, enantioselectively synthesized with the aid of an enzyme. The finding that enantiomer 2 binds to opsin while the other 3 does not defines the absolute sense of twist around the 12-s bond.

Structure and function of chromophores in R-phycoerythrin at 1.9 � resolution

The crystal structure of R-Phycoerythrin (R-PE) from Polysiphonia urceolata has been refined to a resolution of 1.9 A, based on the atomic coordinates of R-PE determined at 2.8 A resolution, through the use of difference Fourier method and steorochemistry parameters restrained refinement with model adjustment according to the electron density map. Crystallographic R-factor of the refined model is 0.195 (Rfree = 0.282) from 8-1.9 A. High resolution structure of R-PE showed precise interactions between the chromophores and protein residues, which explained the spectrum characteristic and function of chromophores. Four chiral atoms of phycourobilin (PUB) were identified as C(4)-S, C(16)-S, C(21)-S, and C(20)-R. In addition to the coupling distances of 19 A to 45 A between the chromophores which were observed and involved in the energy transfer pathway, high resolution structure of R-PE suggested other pathways of energy transfer, such as the ultrashort distance between alpha140a and beta155. It has been proposed that aromatic residues in linker proteins not only influence the conformation of chromophore, but may also bridge chromophores to improve the energy transfer efficiency.

Structure and conformation of the retinal chromophore in bovine rhodopsin.

Structure and conformation of the retinal chromophore in bovine rhodopsin.

Determination of 6s-trans conformation of retinal chromophore in sensory rhodopsin I and phoborhodopsin.

8,16-Ethanoretinal 3 was synthesized from 2-methylcyclohexanone. From the binding experiments of 3 and 8,18-ethanoretinal 2 with apoproteins of sensory rhodopsin I and phoborhodopsin, it was found that retinal was incorporated as 6s-trans conformation in the both proteins.

Enzymatically Synthesized Photodynamic Polyaniline Containing Azobenzene Groups

A novel polyaniline containing azo groups has been synthesized using horseradish peroxidase catalyzed oxidative free radical coupling of 4,4‘-diaminoazobenzene in the presence of hydrogen peroxide. The spectral changes observed indicate polymerization accompanied by trans−cis isomerization, leading to structural constraints in the growing polymer chain. This soluble azo polymer consists of azo groups in the main chain as well as in the side chain. Upon photoexcitation of the polymer in solution, the chromophores undergo structural photoisomerization. The polymer relaxes to a conformation having spectral features identical to those of the conformation prior to photoexcitation if the excitation is carried out within the trans absorption band. Photoexcitation at the cis absorption band or thermal kinetic randomization of the chromophore conformation in the polymer in solution results in relaxation of the structural constraints, which leads to a different constrained conformation.

Conformational analysis of symmetric bilirubin analogues with varying length alkanoic acids. Enantioselectivity by human serum albumin

Symmetric analogues of mesobilirubin-XIIIα, with propionic acid groups shortened to acetic and lengthened to undecanoic, exhibit induced circular dichroism (ICD) in pH 7.5 buffered aqueous [1–5% dimethyl sulfoxide (DMSO) co-solvent] solution in the presence of human serum albumin (HSA). The CD spectra exhibit bisignate Cotton effects with ωIµmax434 = +87, ωIµmax389 = -54 (acetic), ωIµmax436 = +37, ωIµmax388 = -42 (propionic), ωIµmax420 = -15, ωIµmax370 = +8 (butyric), [ωIµmax433 = -97, ωIµmax388 = +89 in 30% aqueous DMSO], ωIµmax449 = +6, ωIµmax397 = -46 (valeric), ωIµmax440 = +57, ωIµmax392 = -96 (caproic), ωIµmax440 = +15, ωIµmax393 = -21 (caprylic) and ωIµmax448 = +18, ωIµmax385 = -31 (undecanoic). These values result from chromophore conformation (i.e. exciton coupling) and enantioselectivity by the protein (i.e. preference for a given bilirubin enantiomer). The UV–VIS spectra of the acetic to butyric, caprylic and undecanoic complexes are similar in shape, with a shoulder in addition to the main band, and reminiscent of that of the bilirubin-IXα HSA complex, indicating an analogous, folded conformation for all. The spectra of the valeric and caproic complexes, in turn, are more symmetric and red-shifted, suggesting a more extended conformation. Experimental CD values in each of these two series have been interpreted in terms of the different enantioselectivity by the protein, with the right handed acetic and caproic enantiomers fitting best the protein binding site (ωωIµ ca. 150) and the protein showing a lower preference for the right handed propionic enantiomer (ωωIµ ca. 80) and even lower for the right handed valeric, caprylic and undecanoic enantiomer (ωωIµ ca. 50), but left handed butyric enantiomer (ωωIµ ca. 24).The differences observed in the UV–VIS spectra of each complexed (in aqueous buffer) vs. uncomplexed pigment (in MeOH), i.e. spectral shifts (7–11 nm for acetic to butyric and undecanoic, 12 nm for valeric and 16–18 nm for caproic and caprylic) and shape (reduction from two to one transition for valeric and caproic—but not for the rest) reflect the changes in pigment conformation induced by the protein. These changes are especially noticeable for the caproic and caprylic analogues.Taken collectively, the present results indicate that the length of the alkanoic acid chains at C8 and C12 is essential for determining not only the pigment conformation, but also the enantioselectivity by the protein (through specific pigment–protein interactions) and agree with previous suggestions that these interactions may involve (at least) one salt linkage and hydrogen bonding.The effect upon the ICD of each rubin-HSA complex of other parameters such as the percentage of DMSO used as carrier in the solution and the nature of the buffer has also been investigated. Surprisingly, an increase in the amount of DMSO from 3–30% results in dramatic changes in the observed CD of the butyric and (to a lesser extent) propionic, undecanoic complexes. These have been interpreted in terms of selective changes in the tertiary structure of the protein.

2H NMR studies of oriented bacteriorhodopsin membranes to determine single bond orientations

AbstractFor the structural analysis of oriented polymers and other macromolecular systems, we present a solid state 2H NMR technique capable of measuring the orientations of individual chemical bond vectors with high accuracy. When an immobilized uniaxially oriented sample is aligned along the spectrometer magnetic field direction, the angle between a deuterium‐labelled methyl group and the axis of ordering can be calculated from the spectral quadrupole splitting. However, since positive and negative splittings are indistinguishable, there may be two solutions. We show how these may be discriminated by acquiring additional spectra at different sample inclinations (Ref 1). The analysis of the resulting complex lineshapes is aided by computer simulation, which furthermore allows a characterization of the orientational distribution function of the sample. The 2H NMR method has been developed around the membrane protein bacteriorhodopsin as a biological model system that can be prepared in uniaxially oriented films. The overall orientation and molecular conformation of the retinal chromophore were resolved from the individual orientations of its five labelled methyl groups (Refs 2, 3). Structural changes were detected in an intermediate of the photocycle (M), which provide insight into the mechanism of the protein as a proton pump (Ref 4).

High-pressure near-infrared Raman spectroscopy of bacteriorhodopsin light to dark adaptation

Near-infrared (NIR) Raman spectroscopy is employed as an in situ probe of the chromophore conformation to study the light to dark-adaptation process in bacteriorhodopsin (bR) at variable pressure and temperature in the absence of undesired photoreactions. In dark-adapted bR deconvolution of the ethylenic mode into bands assigned to the all-trans (1526 cm-1) and 13-cis (1534 cm-1) isomers yields a 13-cis to all-trans ratio equal to 1 at ambient pressure (Schulte et al., 1995, Appl. Spectrosc. 49:80-83). Detailed spectroscopic evidence is presented that at high pressure the equilibrium is shifted toward the 13-cis isomers and that the light to dark adaptation kinetics is accelerated. The change in isomeric composition with temperature and pressure as well as the kinetics support a two-state model activation volumes of -16 ml/mol for the transition of 13-cis to all-trans and -22 ml/mol for the reverse process. These compare with a conformational volume difference of 6.6 ml/mol, which may be attributed to the ionization of one or two residues or the formation of three hydrogen bonds.

Structure Determination of te Biliverdin Apomyoglobin Complex: Crystal Structure Analysis of Two Crystal Forms at 1.4 and 1.5 Å Resolution

Crystal structure determinations of two orthorhombic (P212121) crystal modifications of the biliverdin apomyoglobin complex are described. The two structures were determined by X-ray diffraction at 100 K to a resolution of 1.5 Å and 1.4 Å. Both crystal forms were grown by hanging-drop techniques, using phosphate as precipitant. The structures were solved by molecular replacement and refined to finalR-values of 19.4% and 21.2%.Both structures are very similar with respect to the binding site and the conformation of the biliverdin chromophore, which occurs in a (P) helical conformation. It is located within the heme pocket, very close in position and orientation to the heme binding site in myoglobin. Two water molecules not present in the crystal structure of myoglobin are sequestered within the heme pocket in the biliverdin-apomyoglobin complex, and they are engaged in hydrogen bonding to the biliverdin and to the protein. Comparison with structural results from an earlier NMR study of the same complex shows good agreement.

Synthesis of 11Z-8,18-propano- and methano-retinals and their interaction with bovine opsin.

In order to clarify the conformation of chromophore in rhodopsin, especially the torsional angle around the C6-C7 single bond, 8, 18-propano- and methano-retinals were prepared from 2, 2-dimethylcyclohexanone via the palladium-catalyzed coupling reaction of vinyl triflates with methyl E-3-trimethylstannyl-2-butenoate, and their interaction with bovine opsin was investigated.

A Fourier-transform Raman study of the temperature dependence of chromophore conformation in light-adapted and dark-adapted bacteriorhodopsin

Abstract Fourier-transform Raman spectra are reported for bacteriorhodopsin (BR) in its light-adapted and dark-adapted states at room and elevated temperatures. Qualitative changes are observed in the FT-Raman spectrum above the midpoint temperature of the predenaturational transition indicating structural changes and inhomogeneous protein populations. Dark-adapted BR shows an increased all- trans component at 78°C, while light-adapted BR at 78°C is found to contain 13- cis BR, possibly as a result of a reduced extent of light adaptation or a change in protein—chromophore interactions.

STRUCTURAL DIFFERENCES BETWEEN PHYCOCYANIN and ALLOPHYCOCYANIN FROM THEIR RESONANCE RAMAN SPECTRA*

Abstract— Resonance Raman spectra of the chromophores of the cyanobacterial light‐harvesting proteins phycocyanin (CPC) and allophycocyanin (APC) were recorded using 364 nm excitation. The1500–1700 cm‐1 regions of these spectra were analyzed for the pH‐induced structural changes accompanying the disruption of the native trimers into monomers as well as the progressive denaturation of these monomers. Computer‐assisted decomposition of the 1642 cm‐1 marker bands of these spectra yielded up to four components (named I‐IV), the frequencies of which were constant within 5 cm‐1 (CPC) and 10 cm‐1 (APC). The relative intensities of two of these components, namely I and III, were sensitive to chromophore conformations. The previously reported downshift of the 1642 cm‐1 band upon folding of the chromophores was shown to result from a weakening of component I and a concomitant enhancement of component III. Components I‐IV had different relative intensity patterns in CPC and APC spectra. In particular, the higher relative intensity of component I at 1646 cm‐1 indicated more extended average conformations of the chromophores in trimeric APC than in trimeric CPC. This difference likely resulted from the extra β‐155 chromophore present in CPC. Component III was sizably active in RR spectra of monomelic APC but was not observed either in those of monomelic CPC or in those of trimeric APC and CPC. This indicated that, in APC monomers, chromophore(s) did not assume the native conformations found in the trimer, while monomer formation did not sizably alter the structures of the CPC chromophores.

Deuterium solid-state nuclear magnetic resonance studies of methyl group dynamics in bacteriorhodopsin and retinal model compounds: evidence for a 6-s-trans chromophore in the protein.

Solid-state deuterium NMR spectroscopy is used to examine the dynamic behavior of 18-CD3 methyl groups in microcrystalline 6-s-cis-retinoic acid (triclinic) and 6-s-trans-retinoic acid (monoclinic) model compounds, as well as in the membrane protein bacteriorhodopsin (bR), regenerated with CD3-labeled retinal. Temperature dependent quadrupolar echo line shapes and T1 anisotropy measurements were used to characterize activation energies for 3-fold hopping motion of the methyl groups. These data provide supporting evidence that the conformation of the retinal chromophore in bR is 6-s-trans. The 6-s-cis conformer is characterized by strong eclipsing interactions between the 8-C proton and the 18-C methyl group protons; the 18-CD3 group shows an activation energy barrier for methyl 3-fold hopping of 14.5 +/- 1 kJ/mol. In contrast, the 18-CD3 group in the 6-s-trans isomer shows a considerably lower activation energy barrier of 5 +/- 1 kJ/mol. In bR, it is possible to obtain an approximate activation energy of 9 kJ/mol. This data is inconsistent with a 6-s-cis conformer but is consistent with the existence of a 6-s-trans-retinal Schiff base in bR with some interaction with the protein matrix. These results suggest that methyl rotor motions can be used to probe the van der Waals contact between a ligand and a protein binding pocket. The 6-s-trans conformer of the [16,17-(CD3)2]retinal in frozen hexane exhibits a major kinetic component with an activation energy barrier of of 14 -/+ 2 kJ/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorescence spectra of R-phycoerythrin

The phosphorescence of R-phycoerythrin from the red alga Callithamnion corymbosum was detected. The phosphorescence spectrum of R-phycoerythrin has two main components with maxima at 720 nm and 820 nm and lifetimes of 2.5 ms and 6.0 ms respectively. The phosphorescence excitation spectrum exhibited three bands at 498, 541 and 566 nm, characteristic of the absorption spectrum of R-phycoerythrin, but their relative intensifies differed from those recorded in fluorescence excitation spectrum. The fluorescence quantum yield of the pigment was 0.8 at 77 K; the phosphorescence quantum yield was 1.6 × 10 −6 and it increased to 1.5 × 10 −4 for the R-phycoerythrin denatured with 8 M urea at pH 3.0. This might be explained partly by alteration of the chromophore conformation accompanying the perturbation of protein structure.

Retinoids and related compounds. Part 15. Synthesis and spectral characterization of bicyclic retinals involving the 8–18 or 8–16 bonded structure

In order to investigate both the chromophore conformation around the trimethylcyclohexene ring and the origin of the induced β-circular dichroism (CD) band in rhodopsin, two C(6)–C(7) singlebond fixed retinal analogues, 6s-cis- and 6s-trans-locked bicyclic retinals 5 and 6, have been synthesized. Their spectral characterization is described.