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Difference Spectra (difference + spectrum)
Selected AbstractsCharacterization of the Cph1 holo-phytochrome from Synechocystis sp.FEBS JOURNAL, Issue 7 2001PCC 680 The cph1 gene from the unicellular cyanobacterium Synechoycstis sp. PCC 6803 encodes a protein with the characteristics of plant phytochromes and histidine kinases of two-component phospho-relay systems. Spectral and biochemical properties of Cph1 have been intensely studied in vitro using protein from recombinant systems, but virtually nothing is known about the situation in the natural host. In the present study, His6 -tagged Cph1 was isolated from Synechocystis cells. The cph1,his gene was expressed either under the control of the natural cph1 promoter or over-expressed using the strong promoter of the psbA2 gene. Upon purification with nickel affinity chromatography, the presence of Cph1 in extracts was confirmed by immunoblotting and Zn2+ -induced fluorescence. The Cph1 extracts exhibited a red/far-red photoactivity characteristic of phytochromes. Difference spectra were identical with those of the phycocyanobilin adduct of recombinant Cph1, implying that phycocyanobilin is the chromophore of Cph1 in Synechocystis. [source] The Alzheimer ,-peptide shows temperature-dependent transitions between left-handed 31 -helix, ,-strand and random coil secondary structuresFEBS JOURNAL, Issue 15 2005Jens Danielsson The temperature-induced structural transitions of the full length Alzheimer amyloid ,-peptide [A,(1,40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left-handed 31 (polyproline II; PII)-helix at 0 °C to a random coil state at 60 °C, with an average ,H of 6.8 ± 1.4 kJ·mol,1 per residue, obtained by fitting a Zimm,Bragg model to the CD data. The transition is noncooperative for the shortest N-terminal fragment A,(1,9) and weakly cooperative for A,(1,40) and the longer fragments. By analysing the temperature-dependent 3JHNH, couplings and hydrodynamic radii obtained by NMR for A,(1,9) and A,(12,28), we found that the structure transition includes more than two states. The N-terminal hydrophilic A,(1,9) populates PII-like conformations at 0 °C, then when the temperature increases, conformations with dihedral angles moving towards ,-strand at 20 °C, and approaches random coil at 60 °C. The residues in the central hydrophobic (18,28) segment show varying behaviour, but there is a significant contribution of ,-strand-like conformations at all temperatures below 20 °C. The C-terminal (29,40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of A, may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques. [source] Monitoring of a second-order reaction by electronic absorption spectroscopy using combined chemometric and kinetic modelsJOURNAL OF CHEMOMETRICS, Issue 6 2003Tom J. Thurston Abstract This paper reports the application of 11 methods for obtaining kinetic constants from a second-order reaction, that between phenylhydrazine and benzophenone. In this type of reaction the number of absorbing species is lower than the number of steps in the reaction minus one, resulting in a rank-deficient response matrix. The methods used include traditional univariate curve fitting, classical least squares using previously recorded pure spectra, alternating least squares methods with both kinetic and non-negativity constraints, and target-testing methods using principal component scores. An additional recently proposed method based on difference spectra is also examined, suitable for any single-step closed reaction. The methods that performed best were difference spectra, kinetically constrained alternating least squares, and target-testing approaches. Limitations of the traditional methods are described. Copyright © 2003 John Wiley & Sons, Ltd. [source] Prediction of the association state of insulin using spectral parametersJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2003Vladimir N. Uversky Abstract Human insulin exists in different association states, from monomer to hexamer, depending on the conditions. In the presence of zinc the "normal" state is a hexamer. The structural properties of 20 variants of human insulin were studied by near-UV circular dichroism, fluorescence spectroscopy, and small-angle X-ray scattering (SAXS). The mutants showed different degrees of association (monomer, dimers, tetramers, and hexamers) at neutral pH. A correlation was shown between the accessibility of tyrosines to acrylamide quenching and the degree of association of the insulin mutants. The near-UV CD spectra of the insulins were affected by protein association and by mutation-induced structural perturbations. However, the shape and intensity of difference CD spectra, obtained by subtraction of the spectra measured in 20% acetic acid (where all insulin species were monomeric) from the corresponding spectra measured at neutral pH, correlate well with the degree of insulin association. In fact, the near-UV CD difference spectra for monomeric, dimeric, tetrameric, and hexameric insulin are very distinctive, both in terms of intensity and shape. The results show that the spectral properties of the insulins reflect their state of association, and can be used to predict their oligomeric state. © 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:847,858, 2003 [source] Physical image vs structure relation: part 12 , structure of 2,2,5,5-tetramethyl-dihydro-furan-3-one oxime and its protonated forms through isomerization and NMR spectra,JOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 6 2007Ryszard B. Nazarski Abstract The study of an isomeric A/B mixture of the title oxime 1, by photolytic or thermal E,Z -isomerization and NMR measurement including 1H{1H}-NOE difference spectra, led to assignment of the E configuration to its predominating form A. The 1H/13C data were interpreted in terms of steric overcrowding of both forms, especially of the thermolabile photoproduct B. Four classical (empirical) NMR methods of elucidating the oxime geometry were critically tested on these results. Unexpected vapor-phase photoconversion A,B in the window glass-filtered solar UV and spectroscopic findings on their protonated states were discussed, as well. The kinetically controlled formation of the N- protonated species (Z)- 5+ was proved experimentally. In addition, some 1H NMR assignments reported for structurally similar systems were rationalized (3 and 4) or revised (1 and 7,9) with the GIAO-DFT(B3LYP) and/or GIAO-HF calculational results. Copyright © 2007 John Wiley & Sons, Ltd. [source] The Photoreaction of the Photoactive Yellow Protein Domain in the Light Sensor Histidine Kinase Ppr is Influenced by the C-terminal Domains,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Hironari Kamikubo To study the role of the C-terminal domains in the photocycle of a light sensor histidine kinase (Ppr) having a photoactive yellow protein (PYP) domain as the photosensor domain, we analyzed the photocycles of the PYP domain of Ppr (Ppr-PYP) and full-length Ppr. The gene fragment for Ppr-PYP was expressed in Escherichia coli, and it was chemically reconstituted with p- coumaric acid; the full-length gene of Ppr was coexpressed with tyrosine ammonia-lyase and p -coumaric acid ligase for biosynthesis in cells. The light/dark difference spectra of Ppr-PYP were pH sensitive. They were represented as a linear combination of two independent difference spectra analogous to the PYPL/dark and PYPM/dark difference spectra of PYP from Halorhodospira halophila, suggesting that the pH dependence of the difference spectra is explained by the equilibrium shift between the PYPL - and PYPM -like intermediates. The light/dark difference spectrum of Ppr showed the equilibrium shift toward PYPL compared with that of Ppr-PYP. Kinetic measurements of the photocycles of Ppr and Ppr-PYP revealed that the C-terminal domains accelerate the recovery of the dark state. These observations suggest an interaction between the C-terminal domains and the PYP domain during the photocycle, by which light signals captured by the PYP domain are transferred to the C-terminal domains. [source] Thermal Recovery of Iodopsin from Photobleaching Intermediates,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Yasushi Imamoto The chloride effect on the photobleaching process of iodopsin, a chicken red-sensitive cone visual pigment, was studied in detail by time-resolved low-temperature spectroscopy at ,40°C to ,10°C. Decay-associated difference spectra obtained by kinetic analysis using the singular value decomposition method were composed of spectra of BL-iodopsin, lumiiodopsin, metaiodopsin I, metaiodopsin II and metaiodopsin III, essentially identical to those at room temperature. In each conversion step however, iodopsin was partially regenerated, which is not observed in the bleaching process for other visual pigments or iodopsin at room temperature. Moreover, iodopsin was slowly regenerated from the bleached species. The reverse reactions were completely suppressed by substitution of lyotropic NO3, for Cl,, suggesting that Cl, binding to iodopsin interferes with light-induced cis,trans isomerization of the chromophore. It is likely that the water molecule hydrating Cl, forms the additional hydrogen bond(s), by which the protein conformational change necessary to release this steric hindrance becomes enthalpic. As progress of the bleaching process is a consequence of protein conformational change, it is suppressed at low temperatures, resulting in thermal back-isomerization. [source] Effect of Digitonin on the Rhodopsin Meta I-Meta II Equilibrium,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2005Istvan Szundi ABSTRACT Absorbance difference spectra were recorded from 10 ,s to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20°C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large-scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time-resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin. [source] Water and Carboxyl Group Environments in the Dehydration Blueshift of Bacteriorhodopsin,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 5 2000Robert Renthal ABSTRACT The proton channels of the bacteriorhodopsin (BR) proton pump contain bound water molecules. The channels connect the purple membrane surfaces with the protonated retinal Schiff base at the membrane center. Films of purple membrane equilibrated at low relative humidity display a shift of the 570 nm retinal absorbance maximum to 528 nm, with most of the change occurring below 15% relative humidity. Purple membrane films were dehydrated to defined humidities between about 50 and 4.5% and examined by Fourier transform infrared difference spectroscopy. In spectra of dehydrated-minus-hydrated purple membrane, troughs are observed at 3645 and 3550 cm,1, and peaks are observed at 3665 and 3500 cm,1. We attribute these changes to water dissociation from the proton uptake channel and the resulting changes in hydrogen bonding of water that remains bound. Also, in the carboxylic acid spectral region, a trough was observed at 1742 cm,1 and a peak at 1737 cm,1. The magnitude of the trough to peak difference between 1737 and 1742 cm,1 correlates linearly with the extent of the 528 nm pigment. This suggests that a carboxylic acid group or groups is undergoing a change in environment as a result of dehydration, and that this change is linked to the appearance of the 528 nm pigment. Dehydration difference spectra with BR mutants D96N and D115N show that the 1737,1742 cm,1 change is due to Asp 96 and Asp 115. A possible mechanism is suggested that links dissociation of water in the proton uptake channel to the environmental change at the Schiff base site. [source] Water-protein interactions in the molten-globule state of carbonic anhydrase b: An NMR spin-diffusion studyPROTEIN SCIENCE, Issue 8 2000Victor P. Kutyshenko Abstract We have used the homonuclear Overhauser effect (NOE) to characterize a model protein: carbonic anhydrase B. We have obtained NOE difference spectra for this protein, centering the on-resonance signals either at the methyl-proton or at the water-proton signals. The spin-diffusion spectra obtained as a function of protein concentration and temperature provide direct evidence of much greater protein,water interaction in the molten-globule state than in the native and denatured states. Furthermore, although the protein loses its gross tertiary structure in both the molten-globule and denatured states, it remains almost as compact in its molten-globule state as it is in the native state. The spin-diffusion spectra, obtained as a function of a variable delay time after the saturation pulse, allowed us to measure the relaxation times of several types of proton in the solution. These spectra contain enough information to distinguish between those water molecules solvating the protein and the free ones present as bulk water. [source] Characterization of the CuA center in the cytochrome c oxidase from Thermus thermophilus for the spectral range 1800,500 cm,1 with a combined electrochemical and Fourier transform infrared spectroscopic setupBIOPOLYMERS, Issue 1-2 2004M. Wolpert Abstract In this study we present the electrochemically induced Fourier transform infrared (FTIR) difference spectra of the CuA center derived from the ba3 -type cytochrome c oxidase of Thermus thermophilus in the spectral range from 1800 to 500 cm,1. The mid infrared is dominated by the ,(CO) vibrations of the amide I modes at 1688, 1660, and 1635 cm,1, reflecting the redox-induced perturbation of the predominantly ,-sheet type structure. The corresponding amide II signal is found at 1528 cm,1. In the lower frequency range below 800 cm,1, modes from amino acids liganding the CuA center are expected. On the basis of the absorbance spectrum of the isolated amino acids, methionine is identified as an important residue, displaying C,S vibrations at these frequencies. This spectral range was previously disregarded by protein IR spectroscopists, mainly due to the strong absorbance of the solvent, H2O. With an optimized setup, however, IR is found suitable for structure/function studies on proteins. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004 [source] Assignments and hydrogen bond sensitivities of UV resonance Raman bands of the C8-deuterated guanine ringJOURNAL OF RAMAN SPECTROSCOPY, Issue 9 2002Akira Toyama Isotope-edited Raman spectroscopy, a combination of site-selective isotopic labeling and Raman difference spectroscopy, is a useful method for studying the structure and interaction of individual nucleic acid residues in oligonucleotides. To obtain basic data for applying isotope-edited Raman spectroscopy to guanine residues, we studied the vibrational modes of UV resonance Raman bands of the C8-deuterated guanine ring by examining the wavenumber shifts upon seven isotopic substitutions (2- 13C, 2- 15N, 6- 18O, 7- 15N, 8- 13C, 9- 15N and 1,- 13C). The hydrogen bond sensitivities of the Raman bands were also investigated by comparing the Raman spectra recorded in several solvents of different hydrogen bonding properties. Some of the Raman bands were found to be markers of hydrogen bonding at specific donor or acceptor sites on the guanine ring. The Raman bands, which shift on C8-deuteration, remain in the difference spectrum between the unlabeled and C8-deuterated guanine rings. Among them, a negative peak around 1525 cm,1 and a strong positive/negative peak pair around 1485/1465 cm,1 serve as markers of hydrogen bonding at N7 and C6O, respectively. Another weak positive/negative peak pair around 1025/1040 cm,1 is sensitive to hydrogen bonding at the proton donor sites (N1,H and N2,H2). The applicability of the hydrogen bond markers has been tested by using a 22-mer oligonucleotide duplex containing eight guanine residues and its analog in which a single guanine residue is C8-deuterated. The difference spectrum shows that the hydrogen bonding state of the guanine residue at the labeled position is consistent with the Watson,Crick base pair structure of DNA. Isotope-edited Raman spectroscopy is a useful tool for studying the hydrogen bonding state of selected guanine residues in oligonucleotides. Copyright © 2002 John Wiley & Sons, Ltd. [source] | ||||||||||