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Difference Spectroscopy (difference + spectroscopy)
Kinds of Difference Spectroscopy Selected AbstractsProbing the access of protons to the K pathway in the Paracoccus denitrificans cytochrome c oxidaseFEBS JOURNAL, Issue 2 2005Oliver-M. In recent studies on heme-copper oxidases a particular glutamate residue in subunit II has been suggested to constitute the entry point of the so-called K pathway. In contrast, mutations of this residue (E78II) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78IIQ) or reduce it to about 50% (E78IIA); in the latter case, the mutation causes no drastic decrease in heme a3 reduction kinetics under anaerobic conditions, when compared to typical K pathway mutants. Moreover, both mutant enzymes retain full proton-pumping competence. While oxidized-minus-reduced Fourier-transform infrared difference spectroscopy demonstrates that E78II is indeed addressed by the redox state of the enzyme, absence of variations in the spectral range characteristic for protonated aspartic and glutamic acids at ,,1760 to 1710 cm,1 excludes the protonation of E78II in the course of the redox reaction in the studied pH range, although shifts of vibrational modes at 1570 and 1400 cm,1 reflect the reorganization of its deprotonated side chain at pH values greater than 4.8. We therefore conclude that protons do not enter the K channel via E78II in the Paracoccus enzyme. [source] Functional implications of pigments bound to a cyanobacterial cytochrome b6f complexFEBS JOURNAL, Issue 2 2005Stephan-Olav Wenk A highly purified cytochrome b6f complex from the cyanobacterium Synechocystis sp. PCC 6803 selectively binds one chlorophyll a and one carotenoid in analogy to the recent published structure from two other b6f complexes. The unknown function of these pigments was elucidated by spectroscopy and site-directed mutagenesis. Low-temperature redox difference spectroscopy showed red shifts in the chlorophyll and carotenoid spectra upon reduction of cytochrome b6, which indicates coupling of these pigments with the heme groups and thereby with the electron transport. This is supported by the correlated kinetics of these redox reactions and also by the distinct orientation of the chlorophyll molecule with respect to the heme cofactors as shown by linear dichroism spectroscopy. The specific role of the carotenoid echinenone for the cytochrome b6f complex of Synechocystis 6803 was elucidated by a mutant lacking the last step of echinenone biosynthesis. The isolated mutant complex preferentially contained a carotenoid with 0, 1 or 2 hydroxyl groups (most likely 9- cis isomers of ,-carotene, a monohydroxy carotenoid and zeaxanthin, respectively) instead. This indicates a substantial role of the carotenoid , possibly for strucure and assembly , and a specificity of its binding site which is different from those in most other oxygenic photosynthetic organisms. In summary, both pigments are probably involved in the structure, but may also contribute to the dynamics of the cytochrome b6f complex. [source] A model of a gp120 V3 peptide in complex with an HIV-neutralizing antibody based on NMR and mutant cycle-derived constraintsFEBS JOURNAL, Issue 3 2000Anat Zvi The 0.5, monoclonal antibody is a very potent strain-specific HIV-neutralizing antibody raised against gp120, the envelope glycoprotein of HIV-1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody,peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5,, were studied by NMR and double-mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double-mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light-chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy-chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy-chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light-chain tyrosines were used to dock the peptide into the antibody-combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the ,-hairpin conformation and the type-VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. & Anglister, J. (1999) Nat. Struct. Biol.6, 331,335]. [source] Synthesis and characterization of injectable bioadhesive hydrogels for nucleus pulposus replacement and repair of the damaged intervertebral discJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2010J. Vernengo Abstract Bioadhesive polymers are natural or synthetic materials that can be used for soft tissue repair. The aim of this investigation was to develop an injectable, bioadhesive hydrogel with the potential to serve as a synthetic replacement for the nucleus pulposus of the intervertebral disc or as an annulus closure material. Branched copolymers of poly(N -isopropylacrylamide) (PNIPAAm) and poly(ethylene glycol) (PEG) were blended with poly(ethylene imine) (PEI). This three component injectable system can form a precipitated gel at physiological temperature due to the phase transition of PNIPAAm. The injection of glutaraldehyde into the gel core will adhere the implant to the surrounding tissues. 1H NMR results indicated the successful physical incorporation of PEI into the PNIPAAm-PEG network by blending. In addition, the covalent crosslinking between the amine functionalities on the PEI and the aldehyde functionalities on the glutaraldehyde was verified using FTIR difference spectroscopy. Mechanical characterization of these blends showed a significant increase (p < 0.05) in compressive modulus following glutaraldehyde injection. The in vitro bioadhesive force studies with porcine skin showed a significant increase (p < 0.05) in the mean maximum force of detachment for PNIPAAm-PEG/PEI gels when glutaraldehyde was injected into the gel core. The results of this study indicate that the reactivity between amines and aldehyde functionalities can be exploited to impart bioadhesive properties to PNIPAAm-PEG/PEI copolymers. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010 [source] Synthesis and characterisation of 13C and 15N isotopomers of a 1-acyl-7-nitroindolineJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 9 2001John E. T. Corrie Abstract Efficient methods are described for synthesis of isotopomers of the water-soluble, photolabile 1-acyl-7-nitroindoline 5 with either 13C in the carbonyl of the acyl substituent or 15N in the nitro group. The isotopic incorporations were verified by IR difference spectroscopy coupled with flash photolysis. [source] Fluorescence and Raman spectra on painting materials: reconstruction of spectra with mathematical methodsJOURNAL OF RAMAN SPECTROSCOPY, Issue 10 2006Iacopo Osticioli Abstract SERDS (shift excitation difference spectroscopy) and SSRS (subtracted shifted Raman spectroscopy) methods were applied for fluorescence-background rejection in the Raman spectra of colored materials. These techniques are based on the assumption that the fluorescence contribution can be completely eliminated by subtracting two Raman spectra acquired at two shifted laser excitation frequencies. For the SERDS method a micro-Raman experimental apparatus coupled with a tunable diode laser (central emission at 684 nm) was set up. SSRS measurements were made on a commercial micro-Raman instrument; in this case the shifted spectrum was obtained by moving the spectrometer grating. Raman spectra were then reconstructed by applying the difference deconvolution method that automatically converts the difference signals in Raman peaks through a deconvolution operation. These techniques were tested on two reference colors (ultramarine and 6,6,-dibromoindigotine) and two colored samples of unknown composition (a Pompeian pink powder and a blue paint from a XVII century painting). Fluorescence-background subtraction and the following operation of spectra reconstruction took place successfully with no errors in Raman peaks, width and wavenumber position. In addition, even weak spectral details were revealed favoring the comparison with reference data for a molecular identification. Copyright © 2006 John Wiley & Sons, Ltd. [source] Comparison of Raman spectroscopic methods for the determination of supercooled and liquid water temperatureJOURNAL OF RAMAN SPECTROSCOPY, Issue 8 2005Dubravko Risovi Abstract Raman spectroscopy provides an efficient method for non-contact determination of liquid water temperature with high spatial resolution. It can be also used for remote in situ determination of subsurface water temperature. The method is based on temperature-dependent changes of the molecular OH stretching band in the Raman spectra of liquid water. These in turn are attributed to a decrease in intermolecular hydrogen-bonding interactions with increase in temperature. Here, the results of an experimental study employing three different approaches in the determination of temperature from recorded OH stretching band in the Raman spectra of liquid and supercooled water are presented and discussed. The first two methods are based on deconvolution of the spectral band into Gaussian components whose intensities and associated specific spectral markers are temperature dependent, and the third approach is based on Raman difference spectroscopy (RDS). The presented measurements were conducted on distilled and deionized supercooled and liquid water in the temperature range between ,12.5 and +32.5 °C. The results are compared in terms of linearity of response, sensitivity and accuracy. It is shown that the method based on RDS even in the supercooled temperature range provides better accuracy (the standard deviation from the true temperature is ±0.4 K) and linearity in temperature determination than more complicated methods based on Gaussian deconvolution of the OH stretching band. Copyright © 2005 John Wiley & Sons, Ltd. [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] Fluorescence background suppression in Raman spectroscopy using combined Kerr gated and shifted excitation Raman difference techniquesJOURNAL OF RAMAN SPECTROSCOPY, Issue 4 2002P. Matousek An exceptionally high level of fluorescence rejection from resonance Raman spectra was achieved using a combination of two techniques, namely Kerr gated temporal rejection with shifted excitation Raman difference spectroscopy. The method was able to recover the resonance Raman spectrum from the intense fluorescence background with a signal-to-noise ratio at least 10 times higher than that achievable with either of the two approaches used individually. To demonstrate the effectiveness of the technique we obtained the resonance Raman spectrum of the laser dye rhodamine 6G (1 × 10,3 mol dm,3) in methanol by excitation at 532 nm and measuring under the maximum of fluorescence emission at 560,590 nm. The method reached the photon shot noise limit of the residual fluorescence providing a detection limit for Raman spectra 106 times lower than the original fluorescence intensity in an accumulation time of 800 s. A unique feature of the experiment was the way in which the optical parametric amplifier light source was configured to alternate automatically between the two excitation wavelengths using an optogalvanic mirror arrangement. The ultra-high sensitivity of the combined approach holds great promise for selective probing of complex biological systems using resonance Raman spectroscopy. Copyright © 2002 John Wiley & Sons, Ltd. [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] The allosteric transition in DnaK probed by infrared difference spectroscopy.PROTEIN SCIENCE, Issue 2 2006Concerted ATP-induced rearrangement of the substrate binding domain SBD, substrate binding domain; TR-IR, time resolved infrared spectroscopy; IR, infrared spectroscopy Abstract The biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide-induced cycling of DnaK between substrate-accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the nucleotide-induced absorbance changes in the vibrational spectrum of wild-type DnaK was characterized. To assign the conformation sensitive absorption bands, two deletion mutants (one lacking the C-terminal ,-helical subdomain and another comprising only the N-terminal ATPase domain), and a single-point DnaK mutant (T199A) with strongly reduced ATPase activity, were investigated by time-resolved infrared difference spectroscopy combined with the use of caged-nucleotides. The results indicate that (1) ATP, but not ADP, binding promotes a conformational change in both subdomains of the peptide binding domain that can be individually resolved; (2) these conformational changes are kinetically coupled, most likely to ensure a decrease in the affinity of DnaK for peptide substrates and a concomitant displacement of the lid away from the peptide binding site that would promote efficient diffusion of the released peptide to the medium; and (3) the ,-helical subdomain contributes to stabilize the interdomain interface against the thermal challenge and allows bidirectional transmission of the allosteric signal between the ATPase and substrate binding domains at stress temperatures (42°C). [source] Preparation of active enzyme samples for IR studies of Na+/K+ -ATPaseBIOPOLYMERS, Issue 4-5 2002Detlef Thoenges Abstract In the case of the integral membrane protein Na+/K+ -ATPase, preparation of highly concentrated samples for IR difference spectroscopy often leads to inactivation of the enzyme. Therefore, we compared the activity of Na+/K+ -ATPase using different techniques of sample preparation. The loss of activity can be minimized by cooling the sample to 10°C and by the addition of glycerol and dithiothreitol. The activity of Na+/K+ -ATPase isolated from pig kidney is independent of the protein concentration whereas the enzyme from shark rectal gland is inactivated at concentrations above 1 ,g/,L and is thus unsuitable for IR experiments. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 271,274, 2002 [source] UV resonance Raman study of streptavidin binding of biotin and 2-iminobiotin: Comparison with avidinBIOPOLYMERS, Issue 6 2001John Clarkson Abstract UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin by streptavidin, and the results are compared to those previously obtained from the avidin,biotin complex and new data from the avidin,2-iminobiotin complex. UVRR difference spectroscopy using 244-nm excitation reveals changes to the tyrosine (Tyr) and tryptophan (Trp) residues of both proteins upon complex formation. Avidin has four Trp and only one Tyr residue, while streptavidin has eight Trp and six Tyr residues. The spectral changes observed in streptavidin upon the addition of biotin are similar to those observed for avidin. However, the intensity enhancements observed for the streptavidin Trp Raman bands are less than those observed with avidin. The changes observed in the streptavidin Tyr bands are similar to those observed for avidin and are assigned exclusively to the binding site Tyr 43 residue. The Trp and Tyr band changes are due to the exclusion of water and addition of biotin, resulting in a more hydrophobic environment for the binding site residues. The addition of 2-iminobiotin results in spectral changes to both the streptavidin and avidin Trp bands that are very similar to those observed upon the addition of biotin in each protein. The changes to the Tyr bands are very different than those observed with the addition of biotin, and similar spectral changes are observed in both streptavidin and avidin. This is attributable to hydrogen bond changes to the binding site Tyr residue in each protein, and the similar Tyr difference features in both proteins supports the exclusive assignment of the streptavidin Tyr difference features to the binding site Tyr 43. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 307,314, 2001 [source] | ||||||||||