Primary photoprocesses in oxyblepharismin interacting with its native protein partner

The primary photoprocesses in the photoreceptor for the step-up photophobic response of the light-adapted cells of Blepharisma japonicum (OBIP, OxyBlepharismin bInding Protein) have been studied by ultrafast UV–vis transient spectroscopy. The results are rationalized in terms of heterogeneity of the OBIP sample. Two independent classes of chromoprotein are proposed: a “reactive” species, which presents a specific 680-nm band decaying in 4 and 56 ps and a “non-reactive” one, which behaves like the free chromophore (OxyBP) in solution. A bimolecular photooxidation of OxyBP in the presence of 1,4-benzoquinone was performed to record the absorption spectrum of the OxyBP radical cation. Comparison with reactive OBIP suggests that an electron transfer could be involved in the primary photoprocesses of OBIP and possibly trigger the sensory transduction chain of B. japonicum. In addition, the specificity of the chromophore–protein interaction was investigated by studying the artificial complex that OxyBP forms with human serum albumin (HSA). OxyBP–HSA happens to be spectroscopically much closer to free OxyBP than to OBIP. This highlights the specific nature of the interaction between OxyBP and its native protein partner and further supports the proposal that OBIP is the actual photoreceptor for the photophobic response of B. japonicum.     

Primary photoprocesses involved in the sensory protein for the photophobic response of Blepharisma japonicum

We present new femtosecond transient-absorption and picosecond fluorescence experiments performed on OBIP, the oxyblepharismin-binding protein believed to trigger the photophobic response of the ciliate Blepharisma japonicum. The formerly identified heterogeneity of the sample is confirmed and rationalized in terms of two independent populations, called rOBIP and nrOBIP. The rOBIP population undergoes a fast photocycle restoring the initial ground state in less than 500 ps. Intermolecular electron transfer followed by electron recombination is identified as the excited-state decay route. The experimental results support the coexistence of the oxyblepharismin (OxyBP) radical cation signature with a stimulated-emission signal at all times of the evolution of the transient-absorption spectra. This observation is interpreted by an equilibrium being reached between the locally excited state and a charge-transfer state on the ground of a theory developed by Mataga and co-workers to explain the fluorescence quenching of aromatic hydrogen-bonded donor-acceptor pairs in nonpolar solvents. OxyBP is supposed to bind to an as yet unknown electron acceptor by a hydrogen-bond (HB) and the coordinate along which forward and backward electron transfer proceed is assumed to be the shift of the HB proton. The observed kinetic isotope effect supports this interpretation. Protein relaxation is finally proposed to accompany the whole process and give rise to the highly multiexponential observed dynamics. As previously reported, the fast photocycle of rOBIP can be interpreted as an efficient sunscreen mechanism that protects Blepharisma japonicum from continuous irradiation. The nrOBIP population, the transient-absorption of which strongly reminds that of free OxyBP in solution, might be proposed to actually trigger the photophobic response of the organism through excited-state deprotonation of the chromophore occurring in the nanosecond regime. Additional femtosecond transient-absorption spectra of OxyBP and peri-deprotonated OxyBP are also reported and used as a comparison basis to interpret the results on OBIP.     

Sensitive circular dichroism marker for the chromophore environment of photoactive yellow protein: assignment of the 307 and 318 nm bands to the n --> pi* transition of the carbonyl

The absorption and CD spectra of wild-type PYP, apo-PYP, and the mutants, E46Q and M100A, were measured between 250 and 550 nm. At neutral pH, the two very weak absorption bands of wild-type PYP at 307 and 318 nm (epsilon(max) = 600 +/- 100 M(-1) cm(-1) at 318 nm) are associated with quite strong positive CD bands (Deltaepsilon(max) approximately 6.8 M(-1) cm(-1)). Both sets of bands are absent in the apoprotein. On the basis of this evidence, we assign these optical signals to the n --> pi* transition of the oxygen of the carbonyl group of the 4-hydroxycinnamic acid chromophore, which is expected to be electric dipole forbidden but magnetic dipole allowed. The progression of narrow bands at 307 and 318 nm with a shoulder in the CD around 329 nm is due to vibrational fine structure with a frequency of about 1050 +/- 50 cm(-1). This is the carbonyl stretch frequency in the electronically excited state and is well-known from the vibrational structure in the CD spectra of carbonyl compounds. The positive sign of the CD in the near UV is in accordance with the octant rule and the high-resolution X-ray structure, if we assume that the NH group of cysteine 69 to which the carbonyl is hydrogen bonded is the principle perturbant. Similar absorption and CD spectra were observed in the range of 300-340 nm for the mutants E46Q and M100A at neutral pH. Protonation of the trans chromophore by lowering the pH in the dark (without photoisomerization) broadens the 307 and 318 nm CD bands in the mutant E46Q but does not significantly affect their positions or alter their sign. For the long-lived I(2) photointermediate of the mutant M100A with protonated cis chromophore, we observed that the sign of the rotational strength in the 310-320 nm range is negative (i.e., opposite to that in the dark state with trans chromophore). This suggests that the light-induced isomerization of the chromophore, which leads to breaking of the hydrogen bond with the backbone amide of C69, brings the carbonyl into a new protein environment with different asymmetry than in the unbleached protein. The observed change in sign is mainly due to this effect, but a change in chromophore twist may also contribute. Thus, the 318 nm CD signal is a sensitive marker for the environment of the chromophore carbonyl, which samples various environments and configurations during the photocycle.     

The role of the protein matrix in green fluorescent protein fluorescence

In the ground state of the highly conjugated green fluorescent protein (GFP), the chromophore should be planar. However, numerous crystal structures of GFP and GFP-like proteins have been reported with slightly twisted chromophores. We have previously shown that the protein cavity surrounding the chromophore in wild-type GFP is not complementary with a planar chromophore. This study shows that the crystal structure of wild-type GFP is not an anomaly: most of the GFP and GFP-like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore. When the pi-conjugation across the ethylenic bridge of the chromophore is removed the protein matrix will significantly twist the freely rotating chromophore from the relatively planar structures found in the crystal structures. The possible consequences of this nonplanar deformation on the photophysics of GFP are discussed. A volume analysis of the cis-trans-isomerization of HBDI, a GFP chromophore model compound, reveals that its hula-twist motion is volume conserving. This means that, if the GFP chromophore or GFP chromophore model compounds undergo a cis-trans-isomerization in a volume-constricting medium, such as a protein matrix or viscous liquid, it will probably isomerize by means of a HT-type motion.     

EXCITATION AND FLUORESCENCE SPECTRA OF THE CHROMOPHORE ASSOCIATED WITH THE DNA-PHOTOREACTIVATING ENZYME FROM THE BLUE–GREEN ALGA ANACYSTIS NIDULANS

DNA-PHOTOREACTIVATING enzymes can be classified as deoxyribonucleate cyclobutane dipyrimidine photolyases*. Such an enzyme was recently purified 3760-fold from the blue-green alga Anacystis niduluns [8]. The absorption spectrum of the enzyme revealed a small peak at 418 nm that was attributed to an impurity. The enzyme has now been purified further, by affinity chromatography on far-ultraviolet (far-u.v.) irradiated DNA non-covalently bonded to cellulose, and its excitation and fluorescence spectra measured. These spectra reveal the presence of a non protein chromophore associated with the algal photolyase. The peak wavelengths in the excitation and absorption spectra in the visible region are almost identical and close to that observed in the in vitro photoreactivation action spectrum [8], observations supporting the view that this chromophore is involved as a cofactor in DNA photo reactivation.     

THE BEHAVIOR OF DYE-MUCOPOLYSACCHARIDE COMPLEXES—I. SPECTROSCOPIC STUDIES OF THE SYSTEM CHONDROITIN SULFATE A-ACRIDINE ORANGE

Abstract —Absorption, fluorescence, and circular dichroism (CD) spectra have been obtained for a mucopolysaccharide—cationic dye complex. The dye acridine orange exhibits a blue shift in its absorption maximum and a red shift in its fluorescence maximum when mixed with chondroitin sulfate A in aqueous solution. The spectral shifts of the dye appear to be reversed by addition of salt, divalent cations being more effective than monovalent cations. The complex exhibits induced optical activity in the visible absorption spectrum of acridine orange. The biphasic nature of the CD curve is compatible with interpretations involving chromophore coupling.     

Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress?

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ-radiolysis, we have studied the ROS-induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the ˙OH radical, the protein displays a modified RP-HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Förster resonant energy transfer (FRET) between the single W⁵⁷ and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W⁵⁷ as well as major changes in the protein 3D structure, but show that new fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.     

含蒽醌基双分子膜的电子光谱

用荧光和紫外光谱研究了新合成的含蒽醌(2,6)生色基的单链双亲性分子(ANQU)在水溶液中形成的双分子膜聚集体结构。ANQU在稀水溶液中的吸收光谱比其在乙醇稀溶液中的谱峰有较大的红移；其凝胶态相对于液晶态的吸收谱亦有明显红移。结果表明,ANQU双分子膜中分子的堆积方式是J-聚集,蒽醌生色基以头对尾取向方式排列。变温荧光光谱观察双分子膜中蒽醌生色基的荧光光谱强度和峰位极敏感地受到双分子膜物理状态变化的影响。     

Heteronuclear NMR investigation on the structure and dynamics of the chromophore binding pocket of the cyanobacterial phytochrome Cph1

Structural changes of the chromophore in phytochrome proteins associated with its photocycle are still not fully understood. We use heteronuclear NMR to investigate the conformation and dynamics of the chromophore in the binding pocket of the cyanobacterial phytochrome Cph1. On the basis of distance information obtained from three-dimensional nuclear Overhauser enhancement (3D-NOESY) spectra using the photochemically intact photosensory module of Cph1 we demonstrate that the chromophore is in the ZZZssa form in the P(r) (red absorbing form) state and the ZZEssa form in the P(fr) (far-red absorbing form) state of the protein. While ZZZssa for the P(r) state is in agreement with a recently determined X-ray structure, no comparable information for the P(fr) state of photochemically intact phytochrome has been available up to now. In addition, the chromophore in the binding pocket of Cph1 exhibits a notable mobility, which is distinctly different in the two photostates.     

Probing the decay coordinate of the green fluorescent protein: arrest of cis-trans isomerization by the protein significantly narrows the fluorescence spectra

The fluorescence spectra of the wild-type green fluorescence protein (wt-GFP) and the anionic form of p-hydroxybenzylidenedimethylimidazolone (p-HBDI), which models the protein chromophore, were obtained in the 80-300 K temperature range in glycerol/water solvent. The protein spectra have pronounced and well-resolved vibronic structure, at least at lower temperatures. In contrast, the chromophore spectra are very broad and structureless even at the lowest temperatures. Analysis of the spectra shows that the experimentally observed red-shift of the protein spectrum upon heating is apparently caused by quadratic vibronic coupling of the torsional deformation (TD) of the phenyl single bond of the chromophore to the electronic transition. The broad spectra of the chromophore manifest the contribution of different conformations in the glycerol/water solvent. In particular, the lowest-temperature spectrum reflects the distribution over the same TD coordinate in the excited electronic state, which essentially contributes to the asymmetry of the spectrum. Upon heating, motion along this coordinate leads to a configuration from which the radiationless transition takes place. This narrows the distribution along the TD coordinate, causing a more symmetric fluorescence spectrum. We were able to reconstruct the broad, structureless fluorescence spectra of p-HBDI in glycerol/water solutions at various temperatures by convoluting the original wt-GFP spectra with the function describing the distribution of the transition energies of the p-HBDI chromophore. Thus, both the fluorescence broadening and increase in radiationless transition upon removal of the protein chromophore to bulk solvent are consistent with decay by a barrierless TD of the phenyl single bond.     

Density Functional Study of Tetraphenylporphyrin Long-Range Exciton Coupling

The performance of time-dependent density functional theory (TDDFT) for calculations of long-range exciton circular dichroism (CD) is investigated. Tetraphenylporphyrin (TPP) is used as a representative of a class of strongly absorbing chromophores for which exciton CD with chromophore separations of 50 Å and even beyond has been observed experimentally. A dimer model for TPP is set up to reproduce long-range exciton CD previously observed for a brevetoxin derivative. The calculated CD intensity is consistent with TPP separations of over 40 Å. It is found that a hybrid functional with fully long-range corrected range-separated exchange performs best for full TDDFT calculations of the dimer. The range-separation parameter is optimally tuned for TPP, resulting in a good quality TPP absorption spectrum and small DFT delocalization error (measured by the curvature of the energy calculated as a function of fractional electron numbers). Calculated TDDFT data for the absorption spectra of TPP are also used as input for a ‘matrix method’ (MM) model of the exciton CD. For long-range exciton CD, comparison of MM spectra with full TDDFT CD spectra for the dimer shows that the matrix method is capable of producing very accurate results. A MM spectrum obtained from TPP absorption data calculated with the nonhybrid Becke88–Perdew86 (BP) functional is shown to match the experimental brevetoxin spectrum ‘best’, but for the wrong reasons.     

Characterization of secondary structure of neocarzinostatin apoprotein

Characteristics of the secondary structure of neocarzinostatin apoprotein (apo-NCS) were examined by various means. Gaussian analysis of the Fourier-transform infrared (FT-IR) curve and curve-fitting of the circular dichroism (CD) spectrum for apo-NCS revealed that this peptide was abundant in beta-structures. In the presence of sodium dodecyl sulfate (SDS), the CD bands of NCS originating from phenylalanyl, tyrosyl, and cystinyl residues decreased, indicating a conformational change around the chromophore (CNS-chr). On the other hand, apo-NCS, in the SDS system, showed no change of these bands. We showed that the major parts of the protein moiety consist of beta-structures by measurements of the FT-IR and CD spectra of apo-NCS and a prediction of the secondary structure based on the amino acid sequence of the peptide. It seems that properties of the protein may be important to the hydrophobic interaction between NCS-chr and apo-NCS.     

Single oligomer spectra probe chromophore nanoenvironments of tetrameric fluorescent proteins

When analyzing the emission of a large number of individual chromophores embedded in a matrix, the spread of the observed parameters is a characteristic property for the particular chromophore-matrix system. To quantitatively assess the influence of the matrix on the single molecule emission parameters, it is imperative to have a system with a well-defined chromophore nanoenvironment and the possibility to alter these surroundings in a precisely controlled way. Such a system is available in the form of the visible fluorescent proteins, where the chromophore nanoenvironment is defined by the specific protein sequence. We analyze the influence of the chromophore embedding within this defined protein environment on the distribution of the emission maximum wavelength for a number of variants of the fluorescent protein DsRed, and show that this parameter is characteristic of the chromophore-protein matrix combination and largely independent of experimental conditions. We observe that the chemical changes in the vicinity of the chromophore of different variants do not account for the different distributions of emission maximum positions but that the flexibility of the chromophore surrounding has a dominant role in determining the distribution. We find, surprisingly, that the more rigid the chromophore surrounding, the broader the distribution of observed maximum positions. We hypothesize that, after a thermally induced reorientation in the chromophore surrounding, a more flexible system can easily return to its energetic minimum position by fast reorientation, while in more rigid systems the return to the energetic minimum occurs in a stepwise fashion, leading to the broader distribution observed.     

Towards a correlation of absolute configuration and chiroptical properties of alkyl aryl sulfoxides: a coupled-oscillator foundation of the empirical Mislow rule?

The absorption and circular dichroism (CD) data for a series of alkyl aryl sulfoxides 1-16 of known S configuration have been analyzed. The strong bathochromic effect exerted by the nitro group in the para position of the phenyl sulfoxides indicates that the sulfur atom acts as an electron donor moiety towards the phenyl ring. Such behavior requires a significant 2p(C)-3sp3(S) overlap, and therefore the phenyl (and p-substituted phenyl) sulfoxides 1-12, as well as the 2-naphthyl sulfoxides 15 and 16, must assume a conformation which permits such orbital overlap. The steric effect of the peri hydrogen in 1-naphthyl-substituted compounds 13 and 14 does not allow a conformation of this type, and in these compounds the above-mentioned 2p(C) and 3sp3(S) orbitals are positioned in almost orthogonal planes. This conformational difference is clearly shown by the absorption spectra: compounds 1-12, 15, and 16 show the lowest energy sigma --> sigma* transition of the sulfoxide chromophore at approximately 250 nm, indicating the existence of a conjugated S=O chromophore. In contrast, the corresponding absorption in 13 and 14 occurs at about 200 nm, indicating the presence of an isolated S=O chromophore. The CD spectra of 13 and 14 show a negative, couplet-like feature between 250 and 200 nm. This spectral feature can be interpreted in terms of exciton coupling between the allowed sigma --> sigma* transition of the isolated S=O chromophore at 200 nm and the 1B transition of the naphthalene chromophore. In fact, the Harada-Nakanishi rule predicts a negative CD couplet for an S-configured sulfoxide in the conformation found by UV analysis, as found experimentally. The CD spectrum of 13 is quantitatively reproduced by DeVoe coupled-oscillator calculations, strongly implying that a coupled-oscillator mechanism is operative in determining the optical activity of 13 and 14. This approach has also tentatively been extended to the conjugated sulfoxides 1-12, taking into account the coupling of the benzene chromophore 1La transition with the sigma --> sigma* transition of the S=O chromophore. In this case the Harada-Nakanishi rule also predicts a negative CD couplet for the S-configured sulfoxides, as found experimentally.     

A DETAILED RESONANCE RAMAN STUDY OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

Abstract— Resonance Raman spectra of various M₄₁₂ species associated with the bacteriorhodopsin photocycle have been obtained. These correspond to the two forms observed during the formation of M₄1₂ and the two forms that are observed during its decay in absorption experimeents. We do not see any significant difference between the Raman spectra of any of these forms. We therefore conclude that the differences in these species are due to the differences in the protein structure and not in the chromophore.     

Induced circular dichroism and structural assignment of the cyclodextrin inclusion complexes of bicyclic azoalkanes

The stoichiometries and binding constants of the host-guest complexes between the bicyclic azoalkanes 1-6 and alpha-, beta-, and gamma-cyclodextrins (CDs) and the induced circular dichroism (ICD) of the complexes were analyzed. Assisted by proximity relationships obtained from 2D ROESY NMR spectra, the signs and intensities of the ICD spectra are interpreted in terms of the solution structures (co-conformations) of the CD complexes. The ICD assignments are based on the orientation-intensity ICD rules of Harata and Kodaka, which relate the ICD signs and intensities to the relative orientation of the electric dipole transition moment of the n,pi azo chromophore to the CD axis. The influence of the size of the guest and the host is discussed and the effect of introducing an additional chromophore (either a phenyl or a second azo group) on the ICD spectra is demonstrated.     

Primary events in dim light vision: a chemical and spectroscopic approach toward understanding protein/chromophore interactions in rhodopsin

The visual pigment rhodopsin (bovine) is a 40 kDa protein consisting of 348 amino acids, and is a prototypical member of the subfamily A of G protein-coupled receptors (GPCRs). This remarkably efficient light-activated protein (quantum yield = 0.67) binds the chromophore 11-cis-retinal covalently by attachment to Lys296 through a protonated Schiff base. The 11-cis geometry of the retinylidene chromophore keeps the partially active opsin protein locked in its inactive state (inverse agonist). Several retinal analogs with defined configurations and stereochemistry have been incorporated into the apoprotein to give rhodopsin analogs. These incorporation results along with the spectroscopic properties of the rhodopsin analogs clarify the mode of entry of the chromophore into the apoprotein and the biologically relevant conformation of the chromophore in the rhodopsin binding site. In addition, difference UV, CD, and photoaffinity labeling studies with a 3-diazo-4-oxo analog of 11-cis-retinal have been used to chart the movement of the retinylidene chromophore through the various intermediate stages of visual transduction.     

Circular dichroism and configuration of (−)-1-methyl-1,2,3,4,tetrahydroisoquinoline

In the CD (circular dichroism) spectra of (–)-1-methyl-1,2,3,4, tetrahydroisoquinoline, three CE (Cotton effects) are observed in the 300–200 nm region that are due to the absorption bands of the aromatic chromophore. The synthesis of this substance from (S)(–)--phenylethylamine and the CD spectra confirm its (S)-configuration.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 9, pp. 1238–1240, September, 1987.     

青蒿素及其衍生物的圆二色谱研究

本文通过对青蒿素及其16个衍生物的圆二色谱研究, 首交次确定了青蒿素衍衍生物8、9和17的过氧键的圆二色谱的λ~max和它的△s值。并对青蒿素衍生物的圆二色谱出现类似旋光谱中的“背景曲线”的异常现象作了探讨。     

The use of circular dichroism in the investigation of protein structure and function

Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as alpha-helix and beta-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising molten globule intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.