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1.
Steinhoff HJ 《Biological chemistry》2004,385(10):913-920
Recent developments including pulse and multi-frequency techniques make the combination of site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy an attractive approach for the study of protein-protein or protein-oligonucleotide interaction. Analysis of the spin label side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin label side chains allow the modeling of protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. Structural changes can be detected with millisecond time resolution. Inter- and intra-molecular distances are accessible in the range from approximately 0.5 to 8 nm by the combination of continuous wave and pulse EPR methods. Recent applications include the study of transmembrane substrate transport, membrane channel gating, gene regulation and signal transfer. 相似文献
2.
Conventional electron spin resonance (ESR) spectroscopy of different positional isomers of phosphatidylcholine spin labels (n-PCSL; n=5, 7, 10, 12, 14, and 16) has been used to study micellar dispersions made of poly(ethylene glycol)s-phosphatidylethanolamines (PEGs-PEs) polymer-lipids. Such aggregates are currently used as long circulating drug delivery systems "in vivo." We varied both the hydrocarbon chain length and the polymer size of the polymer-lipids. The dependence of the lipid-chain packing density on temperature and on label position as well as the flexibility and polarity profiles with position of chain labeling have been established for the PEGs-PEs micellar dispersions. The results show both similarity and differences either with common micellar dispersions of single chained lyso-palmitoylphosphatidylcholine (C(16)Lyso-PC) or with lamellar dispersions of double chained dipalmitoylphosphatidylcholine (DPPC). Well defined chain flexibility gradients of the same overall shape are obtained in the considered dispersions. However, the mobility of the first acyl chain segments is appreciable higher in micelles of polymer-lipids than in bilayers of DPPC and it becomes indistinguishable at the chain termini. A trend of decreasing polarity on moving toward the bilayer interior is seen in DPPC bilayers, whereas biphasic polarity profiles are obtained in micelles of polymer-lipids and C(16)Lyso-PC. Moreover, the properties of the PEGs-PEs micelles do not depend on the length of the hydrocarbon chain of the polymer-lipids but are slightly influenced by the size of the polymer. 相似文献
3.
Second derivative spectroscopy was used to determine the percentage of tyrosine residues that are exposed to solvent in cytochrome P-450cam isolated from Pseudomonas putida. The ratio between two peak to trough second derivative absorbance differences has been shown to be dependent on the polarity of the microenvironment surrounding tyrosine residues [Ragone, R., Colonana, G., Balestrieri, C., Servillo, L., & Irace, G. (1984) Biochemistry 23, 1871]. With a number of camphor analogues that independently vary the spin equilibrium of the ferric cytochrome P-450 cam, experiments have demonstrated that the percentage of tyrosine residues exposed to solvent is linearly dependent on the percentage of ferric high-spin species present. This is not simply a function of the extent of substrate binding since in all cases the substrate concentration was sufficient to ensure saturation of the cytochrome. The local microenvironment of approximately one tyrosine residue appears to be linearly correlated with the percentage of ferric high-spin cytochrome. Structural studies of cytochrome P-450cam using small-angle X-ray scattering [Lewis, B. A., & Sligar, S. G. (1983) J. Biol. Chem. 258, 3599] and high-pressure difference spectroscopy [Fisher, M. T., Scarlata, S. F., & Sligar, S. G. (1985) Arch. Biochem. Biophys. 240, 456] imply that global conformational changes linked to the spin equilibria are small. Together with the data reported herein, these results suggest that one tyrosine residue is involved in a conformational change that is directly linked with the spin equilibrium. 相似文献
4.
G. Bruce Birrell Terry D. Lee O. Hayes Griffith John F. W. Keana 《Bioorganic chemistry》1978,7(4):409-420
A general synthesis of chlorophyll-derived spin labels is reported. The starting point is chlorophyll a and a long chain spin-labeled alcohol. The four-step synthetic route yields new spin labels in quantities practical for membrane spin-labeling studies. Two examples prepared are 12′-proxyltridecyl pyropheophorbide a and 12′-proxylhexadecyl pyropheophorbide a. The spin labels were purified and characterized by thin-layer chromatography, high-pressure liquid chromatography, mass spectroscopy, nmr and visible spectroscopy, and the number of unpaired spins per molecule. ESR spectral parameters are reported. The chlorophyll-derived spin labels intercalate into fluid phospholipid bilayers and are observed in both the bilayer phase and bound to membrane proteins in chromatophores of the purple photosynthetic bacterium, Rhodopseudomonas sphaeroides. 相似文献
5.
Bartucci R Erilov DA Guzzi R Sportelli L Dzuba SA Marsh D 《Chemistry and physics of lipids》2006,141(1-2):142-157
Recently, developments in time-resolved spin-label electron spin resonance (ESR) spectroscopy have contributed considerably to the study of biomembranes. Two different applications of electron spin echo spectroscopy of spin-labelled phospholipids are reviewed here: (1) the use of partially relaxed echo-detected ESR spectra to study the librational lipid-chain motions in the low-temperature phases of phospholipid bilayers; (2) the use of electron spin echo envelope modulation spectroscopy to determine the penetration of water into phospholipid membranes. Results are described for phosphatidylcholine bilayer membranes, with and without equimolar cholesterol, that are obtained with phosphatidylcholine spin probes site-specifically labelled throughout the sn-2 chain. 相似文献
6.
Identifying conformational changes with site-directed spin labeling 总被引:16,自引:0,他引:16
Site-direct spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for detecting structural changes in proteins. This review provides examples that illustrate strategies for interpreting the data in terms of specific rearrangements in secondary and tertiary structure. The changes in the mobility and solvent accessibility of the spin label side chains, and in the distances between spin labels, report (i) rigid body motions of alpha-helices and beta-strands (ii) relative movements of domains and (iii) changes in secondary structure. Such events can be monitored in the millisecond time-scale, making it possible to follow structural changes during function. There is no upper limit to the size of proteins that can be investigated, and only 50-100 picomoles of protein are required. These features make site-directed spin labeling an attractive approach for the study of structure and dynamics in a wide range of systems. 相似文献
7.
8.
Summary Permeabilities for a homologous series of amine and carboxylate nitroxide spin probes were measured in human red blood cells by an electron paramagnetic resonance (EPR) method. Permeabilities determined in this study are much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. This suggests that the rate-limiting region for permeation of these nonelectrolytes is somewhere in the membrane periphery rather than in the center of the membrane. The red cell membrane does not discriminate between these probes on the basis of molecular volume, as might be predicted by a simple free-volume theory of membrane permeation. 相似文献
9.
M Laloraya 《Biochemical and biophysical research communications》1990,167(2):561-567
The endometrial phospholipid bilayer is shown to be in a highly fluid and polar state at the time of implantation in Mus musculus as evidenced from a spin label study using the stearic acid spin label, 5-doxyl stearate. The positive correlation observed between the superoxide anion radical levels and the fluidity and polarity state of the endometrial cells points towards the possibility of the mediation of this condition of unsaturation of fatty acids to be an act of the superoxide anion radical. 相似文献
10.
An electron spin resonance study of synaptosome opiate receptors. The preparation and use of a spin labeled morphine.
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Morphine spin labeled on the phenolic hydroxy group has been prepared using commercially available reagents and characterized by thin layer chromatography, mass spectroscopy, and electron spin resonance spectroscopy. It has been shown that morphine modified in this way retains some opiate activity, does not pass through the blood-brain barrier, and specifically binds to isolate rat brain synaptosomes. Spin labeled morphine has been shown to be an effective biophysical probe complementing radioactive tracer techniques in the study of the narcotic receptor site. 相似文献
11.
Iva Šnyrychová 《Free radical biology & medicine》2010,48(2):264-274
Electron paramagnetic resonance (EPR) spin trapping spectroscopy is an important method used in free radical research; however, its application in biological systems is hindered by EPR silencing of spin adducts. Previous studies in superoxide-generating chemical systems have shown that spin adducts can be partially stabilized by cyclodextrins. In this work, for the first time, this proposed protective effect of cyclodextrins is investigated in a real biological sample—in isolated thylakoid membranes and photosystem II (PSII) particles with EMPO as a spin trap. It is shown that (i) randomly methylated β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin form inclusion complexes with EMPO–superoxide adducts (EMPO-OOH), (ii) both cyclodextrins increase the intensity of the EMPO-OOH EPR signal in PSII particles up to five times, (iii) higher EMPO-OOH EPR signal intensity is a result of increased stability of EMPO-OOH, and (iv) the extent of the protection of EMPO-OOH adduct provided by cyclodextrins is different in thylakoids and PSII particles. Along with the spin trapping data, the toxicity of cyclodextrins is also discussed with particular focus on photosynthetic preparations. The presented data show that both tested cyclodextrins can be used as valuable tools to improve the sensitivity of spin trapping in biological samples. 相似文献
12.
Joachim Bargon 《Photochemical & photobiological sciences》2006,5(10):970-978
Triplet energies play a considerable role in optical spectroscopy, and can be determined from phosphorescence or the quenching thereof. Their role in spin chemistry may not be as obvious, but the triplet state has always had an important function or utility, namely of reaction intermediates such as radical pairs, their precursors, of carbenes, and of the final products. In situ NMR spectroscopy represents a useful tool to explore certain properties of the triplet state, especially in cases with no phosphorescence. The 'phase' of CIDNP resonances, i.e., emission or enhanced absorption, reflects the spin selectivity of electron transfer reactions. In radical ion pairs the spin selectivity is determined by the relation between the change of the standard free enthalpy DeltaG degrees during the electron back transfer and the triplet energies (E(T)) of the products. If triplet recombination is energetically feasible (DeltaG degrees > E(T)), it is typically the more efficient process in agreement with the Marcus theory. 相似文献
13.
Shigeaki Nakazawa Kazunobu Sato Daisuke Shiomi M. Luisa T.M.B. Franco M. Celina R.L.R. Lazana M. Candida B.L. Shohoji Koichi Itoh Takeji Takui 《Inorganica chimica acta》2008,361(14-15):4031-4037
C60-based polyanionic high-spin clusters (S = 1–3) in their ground state have been prepared by successive chemical reductions of pristine C60 fullerene with potassium in the presence of dicyclohexano-18-crown-6-ether in solution. Intermolecular spin-triplet, quintet, and septet states arising from the C60-based polyanionic molecular clusters have been generated at ambient temperature and identified by CW-ESR and pulse-ESR-based two-dimensional (2D) electron spin transient nutation (2D-ESTN) spectroscopy in organic rigid glasses, for the first time. Intermolecular exchange interactions between mono- and polyanionic C60 fullerenes are ferromagnetic via bridging potassium metal cations. The molecular structures of the polyanionic high-spin C60 clusters in solution have been proposed by a well-established phenomenological spin Hamiltonian approach in terms of the D-tensor-based calculations for the high-spin clusters. The findings of the C60-based high-spin molecular clusters evidence the occurrence of an intramolecular triplet C60 dianion in the ground state. Unequivocal spin identification for molecular high-spin entities by 2D-ESTN spectroscopy and its powerfulness have been illustrated, emphasizing that the 2D-ESTN spectroscopy is useful for mixtures of molecular species with different spin multiplicities, characterized by a small g-anisotropy, for which the powerfulness of advanced high-frequency ESR spectroscopy is hampered. 相似文献
14.
250-GHz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes. 总被引:3,自引:2,他引:1
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Rigid-limit 250-GHz electron spin resonance (FIR-ESR) spectra have been studied for a series of phosphatidylcholine spin labels (n-PC, where n = 5, 7, 10, 12, 16) in pure lipid dispersions of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as well as dispersions of DPPC containing the peptide gramicidin A (GA) in a 1:1 molar ratio. The enhanced g-tensor resolution of 250-GHz ESR for these spin labels permitted a careful study of the nitroxide g-tensor as a function of spin probe location and membrane composition. In particular, as the spin label is displaced from the polar head group, Azz decreases and gxx increases as they assume values typical of a nonpolar environment, appropriate for the hydrophobic alkyl chains in the case of pure lipid dispersions. The field shifts of spectral features due to changes in gxx are an order of magnitude larger than those from changes in Azz. The magnetic tensor parameters measured in the presence of GA were characteristic of a polar environment and showed only a very weak dependence of Azz and gxx on label position. These results demonstrate the significant influence of GA on the local polarity along the lipid molecule, and may reflect increased penetration of water into the alkyl chain region of the lipid in the presence of GA. The spectra from the pure lipid dispersions also exhibit a broad background signal that is most significant for 7-, 10-, and 12-PC, and is more pronounced in DPPC than in POPC. It is attributed to spin probe aggregation yielding spin exchange narrowing.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
D Morikis P M Champion B A Springer K D Egebey S G Sligar 《The Journal of biological chemistry》1990,265(21):12143-12145
We have used resonance Raman spectroscopy to study 11 distal pocket mutants and the "wild type" and native ferric sperm whale myoglobin. The characteristic Raman core-size markers v4, v3, v2, and v10 are utilized to assign the spin and coordination state of each sample. It is demonstrated that replacements of the distal and proximal histidines can discriminate against H2O as a sixth ligand and favor a pentacoordinate Fe3+ atom. Soret absorption band blueshifts are correlated with the pentacoordinate heme environment. One E7 replacement (Arg) leads to an iron spin state change and produces a low spin species. The Glu and Ala mutations at position E11 leave the protein's spin and coordination unaltered. A laser-induced photoreduction effect is observed in all pentacoordinate mutants and seems to be correlated with the loss of the heme-bound water molecule. 相似文献
16.
Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch. 相似文献
17.
P D Hardman 《European journal of biochemistry》1982,124(1):95-101
The thermotropic behaviour of egg yolk phosphatidylethanolamine dispersions in excess aqueous phase has been investigated by spin label electron spin resonance spectroscopy and differential thermal analysis. Phosphatidylethanolamine isomers spin-labelled at six different positions along the acyl chain, and steroid spin labels, indicate both gel-fluid lamellar and lamellar-reverse hexagonal (HII) phase transitions, in agreement with complementary calorimetric studies. Analysis of spin label data shows that the transition to the HII phase is accompanied by an increase in conformational freedom of the acyl chain, more pronounced towards the methyl terminus, and representing an increase in the population of gauche isomers which can only be accommodated by a transition to the non-bilayer phase. Raising the bulk pH to, and above, pH 8.5 results in stabilisation of the bilayer phase and no transition to the HII phase is observed. The phosphatidylethanolamine spin labels also indicate a polarity profile which is characteristic of each phase. 相似文献
18.
R Owenius M Osterlund M Lindgren M Svensson O H Olsen E Persson P O Freskgrd U Carlsson 《Biophysical journal》1999,77(4):2237-2250
Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins. 相似文献
19.
A combination of selective spin decoupling, two-dimensional double quantum spectroscopy, correlated spectroscopy (COSY), and pH titration experiments brought about the assignment of all tyrosyl spin systems and completed the assignment of the histidyl spin systems in porcine adenylate kinase. In the detection of the tyrosyl spin systems it proved to be advantageous to resort to the COSY method rather than to two-dimensional double quantum spectroscopy. In the titration experiments, His189 revealed a second apparent pK value at pH 8.3, which is explained by deprotonation of the adjacent residue Cys187. None of the seven tyrosyl side-chains shows any evidence for deprotonation up to the point of denaturation of the protein, which took place around pH 10. 相似文献
20.
The ultimate goal of in vivo electron spin resonance (ESR) spin trapping is to provide a window to the characterization and quantification of free radicals with time within living organisms. However, the practical application of in vivo ESR to systems involving reactive oxygen radicals has proven challenging. Some of these limitations relate to instrument sensitivity and particularly to the relative stability of these radicals and their nitrone adducts, as well as toxicity limitations with dosing. Our aim here is to review the strengths and weaknesses of both traditional and in vivo ESR spin trapping and to describe new approaches that couple the strengths of spin trapping with methodologies that promise to overcome some of the problems, in particular that of radical adduct decomposition. The new, complementary techniques include: (i) NMR spin trapping, which monitors new NMR lines resulting from diamagnetic products of radical spin adduct degradation and reduction, (ii) detection of *NO by ESR with dithiocarbamate: Fe(II) "spin trap-like" complexes, (iii) MRI spin trapping, which images the dithiocarbamate: Fe(II)-NO complexes by proton relaxation contrast enhancement, and (iv) the use of ESR to follow the reactions of sulfhydryl groups with dithiol biradical spin labels to form "thiol spin label adducts," for monitoring intracellular redox states of glutathione and other thiols. Although some of these approaches are in their infancy, they show promise of adding to the arsenal of techniques to measure and possibly "image" oxidative stress in living organisms in real time. 相似文献