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At least 1,189 records · Page 66Linked to original sources
PubMed1977

A study of conformational changes in two beta-93 modified hemoglobin A's using a triphosphate spin label.

The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.

Diphosphoglyceric Acids↗
PubMed2005

Spatially-confined arterial spin-labeling with FAIR.

PURPOSE: To investigate the effectiveness of slab-selective inversion in pulsed arterial spin labeling with body coil excitation as a means to reduce large vessel contamination of the perfusion signal. MATERIALS AND METHODS: Studies were conducted by varying the tagging width in multislice flow-sensitive alternating inversion recovery (FAIR) in conjunction with body coil excitation on a Siemens Sonata whole-body 1.5-T scanner. The results of spatially-confined tagging were then compared with conventional nonselective tagging in the presence and absence of a bipolar gradient crusher pair in order to determine the effectiveness of suppressing vascular signal and to estimate the bolus width that reaches the capillary bed. RESULTS: It is shown in five volunteers, ages 23-38 years, that depending on the average velocity of the arterial blood flow in the tagging region, a bolus of 6-8 cm in width reaches the capillary bed at a fixed inversion time TI of 1.4 seconds, while a bolus of 11.2-16.5 cm in width enters the imaging region. Further, noticeable velocity differences have been found among the participating subjects, with averages ranging from 10.1 to 13.9 cm/second. CONCLUSION: The data suggest that it is advantageous to replace nonselective global tagging in FAIR perfusion imaging with body coil excitation by spatially-confined tagging to reduce undesired residual tagged blood in large vessels.

Adult↗
PubMed1982

Study on the interaction between hemoglobin Izu (Macaca) and organic phosphates by spin labeling.

ESR spectra of the carbonmonoxy, oxy, and deoxy derivatives of hemoglobin Izu [Hb Izu (Macaca): beta 83 (EF 7) Gly leads to Cys] labeled at cysteine beta 83 with maleimide spin label have been observed in the presence and absence of 2,3-diphosphoglycerate and inositol hexaphosphate. The tau c values obtained from the spectra indicated that inositol hexaphosphate binds to all the derivatives of Hb Izu, but 2,3-diphosphoglycerate only to the deoxy derivatives.

Animals↗
PubMed1992

Luminescence quenching by nitroxide spin labels in aqueous solution: studies on the mechanism of quenching.

The mechanism of luminescence quenching by spin labels was investigated in aqueous solution by steady-state and time-resolved luminescence techniques. Water-soluble nitroxide radicals strongly quenched the luminescence emitted by Tb3+ chelates and by fluorescein, either free or conjugated to proteins. The following features of the quenching reaction were established: (I) the rate constant for quenching of triplet-state Tb3+ by nitroxides was about 4 orders of magnitude smaller (ca. 10(5) M-1 s-1) than those of the singlet-state probes; (II) the quenchers reduced the excited-state lifetime of both probes; (III) the rate constants for quenching of both probes were found to be apparently independent of the temperature (between 6 and 42 degrees C) and viscosity (up to 60 mPa.s) of the solutions; (IV) both singlet and triplet quenching rates were sensitive to solvent polarity; (V) there is a small but significant spectral overlap between the absorption band of weekly absorbing nitroxide radicals and the emission spectra of luminophores, the extent of which, however, does not correlate with the extent of quenching; (VI) the quenching rate declines sharply with an increasing luminophore to nitroxide distance. The distance dependence of the quenching rate showed a satisfactory fit to an exponential function. These findings indicate that the quenching reaction is dominated by an electron exchange between the excited singlet- or triplet-state luminophore and the nitroxide radical rather than controlled by diffusional properties of the reactants.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic N-Oxides↗
PubMed1984

Binding of a spin-labelled chlorpromazine analogue to calmodulin.

The binding of a spin-labelled derivative of chlorpromazine to calmodulin was investigated by e.s.r. spectrometry. The completion of the spectroscopic changes requires the presence of 4 Ca2+ ions per calmodulin molecule. The influences of various physicochemical factors (pH, ionic strength) are discussed in relation to the nature (hydrophobic and polar) of the interactions that hold the drug-calmodulin complex together.

Calcium↗
PubMed2003

Detergent resistant domains in erythrocyte membranes survive after cell cholesterol depletion: an EPR spin label study.

We use electron paramagnetic resonance (EPR) with liposoluble spin labels in order to study the lipid structures obtained after Triton X-100 extraction of erythrocyte membranes. The apparent order profile in these detergent resistant membranes (DRM) is very similar to that of the parent membrane, although with higher absolute values, consistent with a liquid-ordered state. DRM could also be obtained from erythrocytes previously depleted in a 40% of their membrane cholesterol, in apparent opposition to the phenomenon of raft disruption reported by other authors. However, the protein profile of these samples showed important differences with that of DRM from untreated cells. The analysis of our results suggests that the effect of Triton X-100 on cholesterol depleted erythrocytes is limited to the solubilization of raft proteins, without disrupting the lipid matrix of DRM.

Cholesterol↗
PubMed1999

QUIPSS II with thin-slice TI1 periodic saturation: a method for improving accuracy of quantitative perfusion imaging using pulsed arterial spin labeling.

Quantitative imaging of perfusion using a single subtraction, second version (QUIPSS II) is a pulsed arterial spin labeling (ASL) technique for improving the quantitation of perfusion imaging by minimizing two major systematic errors: the variable transit delay from the distal edge of the tagged region to the imaging slices, and the contamination by intravascular signal from tagged blood that flows through the imaging slices. However, residual errors remain due to incomplete saturation of spins over the slab-shaped tagged region by the QUIPSS II saturation pulse, and spatial mismatch of the distal edge of the saturation and inversion slice profiles. By replacing the original QUIPSS II saturation pulse with a train of thin-slice periodic saturation pulses applied at the distal end of the tagged region, the accuracy of perfusion quantitation is improved. Results of single and multislice studies are reported.

Brain↗
PubMed2001

Time-resolved detection of transient movement of helices F and G in doubly spin-labeled bacteriorhodopsin.

Photo-excited structural changes of the light-driven proton pump bacteriorhodopsin were monitored using double-site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy. The inter-spin distances between nitroxides attached at residue positions 100 and 226, 101 and 160, and 101 and 168 were determined for the BR initial state and the trapped M photo-intermediate. Distance changes that occur during the photocycle were followed with millisecond time resolution under physiological conditions at 293 K. The kinetic analysis of the EPR data and comparison with the absorbance changes in the visible spectrum reveal an outward movement of helix F during the late M intermediate and a subsequent approach of helix G toward the proton channel. The displacements of the cytoplasmic moieties of these helices amount to 0.1-0.2 nm. We propose that the resulting opening of the proton channel decreases the pK of the proton donor D96 and facilitates proton transfer to the Schiff base during the M-to-N transition.

Bacteriorhodopsins↗
PubMed1975

Calcium-induced conformational changes and mutual interactions of troponin components as studied by spin labeling.

The three troponin components, TN-C, TN-I, and TN-T, were spin-labeled with two different derivatives of the nitroxide radical, a maleimide and an imidazole reagent. The ESR spectra of various combinations of labeled and unlabeled components were measured both in the presence and absence of calcium. Conformational changes due to the binding of the components and also due to the binding of calcium were sensitively detected in many combinations as large changes in the spectrum. The conformation of TN-C was modified by both TN-T and TN-I. The effects were larger in the presence of calcium than in its absence. In the presence of calcium, TN-T and TN-I both showed large effects with the maleimide label, while TN-I showed a larger effect than TN-T with the imidazole label. In the absence of calcium, the effect of TN-I was larger than that of TN-T. The senstivitiy of TN-C to calcium was magnified by component binding, since the conformation of TN-C itself was not greatly affected by calcium. The conformation of TN-I was greatly altered only in the presence of both TN-C and calcium. This indicates that the calcium-induced conformational change in TN-C is transmitted to the adjacent TN-I. In reconstituted troponin, the conformation of TN-C was more influenced by TN-I in the presence of calcium and by TN-T in its absence as indicated by the imidazole label. With the maleimide label, TN-I was more influential in the absence of calcium. The effect of calcium on the troponin complex was to make the local environment of the label more rigid. The half-maximal effect was observed at 2 X 10(-6)M calcium with TN-I in various complexes, while it was 10(-5)M with TN-C in the complexes. In any case the calcium effects became discernible at 10(-6)M and saturated at 10(-4)M.

Animals↗
PubMed1983

Non-uniform distribution of phospholipids in (Na+ + K+)-ATPase-rich membranes from Torpedo marmorata electric organ evidenced by spin-spin interactions between spin-labeled phospholipids.

(Na+ + K+)-ATPase membranes from Torpedo marmorata electric organ were labeled with high concentrations of paramagnetic phospholipid analogs (up to 5 mol of spin labels for 100 mol of endogeneous phospholipids). The mobile lipid bilayer component of the complex resonance spectra obtained showed low-field linewidth broadening due to spin-spin interactions; this was used as a measure of probe concentration in this compartment. Our results show that in the lipid shell surrounding the protein, there is a considerable enrichment in negatively charged phospholipids over neutral ones.

Animals↗
PubMed1987

Synthesis of spin-labeled photoaffinity derivatives of NAD+ and their interaction with lactate dehydrogenase.

The synthesis of NAD+ derivatives spin-labeled at either N6 or C8 of the adenine ring is described, in which the carboxamide function of the nicotinamide moiety is replaced by a diazirine ring. Irradiation of these compounds at 350 nm generates a carbene which will react with any functional group in its vicinity including hydrocarbons. Both NAD+ derivatives form tight ternary complexes with lactate dehydrogenase and were covalently incorporated into this enzyme. They may be employed for ESR studies when non-covalent interactions are too weak for motionally restricted species to be observed.

Animals↗
PubMed2003

Susceptibility contrast and arterial spin labeled perfusion MRI in cerebrovascular disease.

PURPOSE: To directly compare dynamic susceptibility contrast (DSC) and continuous arterial spin labeled (CASL) magnetic resonance (MR) perfusion techniques in patients with known cerebrovascular disease, with the goals of identifying possible pitfalls in interpretation and determining potential for a complementary role in this setting. METHODS: DSC and CASL MR perfusion studies were performed and compared in 11 patients with acute and/or chronic cerebrovascular disease. Using an automated segmentation technique, Pearson correlation coefficients were generated for CASL perfusion measurements compared to DSC perfusion maps (time-to-peak [TTP], relative cerebral blood volume [rCBV], cerebral blood flow [rCBF], and mean transit time [MTT]) by hemisphere and vascular territory. RESULTS: TTP maps obtained using DSC perfusion MR correlated best both subjectively and objectively with CASL perfusion MR measurements when all patients studied were considered. If patients with a major transit delay were excluded, DSC rCBF correlated best with CASL CBF measurements. CONCLUSION: There may be a complementary role for CASL and DSC perfusion MR methods in cerebrovascular disease, especially in the setting of a marked transit delay.

Acute Disease↗
PubMed1984

Motional dynamics of a spin labeled substrate analogue bound to cytochrome P-450: saturation transfer EPR studies.

The rotational motion of the spin labeled substrate analogue n- propylisocyanide bound to the active center of cytochrome P-450 was studied by saturation transfer EPR. The observed motional rate characterized by an effective rotational correlation time tau R of about 40 ns at 20 degrees C is at least 3 orders of magnitude higher than the macromolecular rotational diffusion of cytochrome P-450 in the microsomal membrane and represents a considerable motion in relation to the whole enzyme molecule. The tau R value is independent on the degree of purification of the enzyme system as revealed by measurements of (1) liver microsomes, (2) partially purified cytochrome P-450, and (3) cytochrome P-450 LM2 but shows a characteristic temperature dependence in the case of microsomes resulting in breaks in the Arrhenius plot at temperatures which correspond to phase transitions of the phospholipids. The results indicate that the mobility of the bound substrate analogue reflecting a relatively high conformational flexibility of the substrate binding region which depends on the state of the lipids and can therefore be influenced by them. These results support the assumption that cytochrome P-450 is capable of forming manifold binding to substrate molecules differing in stereochemical structures because of the conformational flexibility of its binding region.

Animals↗
PubMed1977

Membrane-controlled depletion of complement activity by spin-label-specific IgM.

Complement depletion mediated by high molecular weight (IgM) rabbit antibodies specifically bound to spin-label lipid haptens dispersed in model membranes is controlled by various physical attributes of those membranes other than the total number of exposed determinants that they provide. Carrier lipids used at 32 degrees were (i) a "fluid" phosphatidylcholine (PC), (ii) a "solid" PC, and (iii) a cholesterol/PC mixture. The concentration of hapten in the plane of the membranes (two-dimensional concentration) was varied while the overall hapten molarity (three-dimensional concentration) was kept constant. Both specific binding and the efficiency of depletion by IgM are markedly enhanced by systematically decreasing the average distance between haptens (infinity --> 26 A). Heterogeneous distribution was found to be more favorable than a random homogeneous distribution of the same number of haptens in the same total quantity of lipids. IgM efficiency is also markedly increased by the inclusion of cholesterol in PC membranes, an effect thought to result from enhanced projection of the determinant from the surface of the membrane and hence increased accessibility to the antibody-binding site. Furthermore, the efficiency of IgM was increased by using haptens dispersed in fluid rather than in solid PC membranes. The results are consistent with the hypothesis that IgM molecules must be bound to a critical multiple of antigenic determinants at a membrane surface in order to induce complement-mediated attack and that subtle variation of the physical state of membrane antigens can be the crucial factor in determining the outcome of this type of efferent immune response.

Animals↗
PubMed1983

Saturation transfer EPR spectroscopy on spin-labeled muscle fibers using a loop-gap resonator.

Previously, saturation transfer (ST-EPR) studies of biomolecular dynamics have involved the use of a resonant cavity and the V'2 display (absorption, second harmonic, out of phase). In the present study, we replaced the resonant cavity with a loop-gap resonator and used the U'1 display (dispersion, first harmonic, out of phase) to study spin-labeled muscle fibers. The new resonator and display showed several advantages over those previously used. It produced virtually noiseless U'1 spectra on a 0.4 microliter sample using a 4 min scan; previous U'1 experiments on spin-labeled muscle, using a conventional rectangular cavity, resulted in an unacceptably low signal-to-noise ratio. The high filling factor of the resonator facilitated the study of these extremely small fiber bundles and permitted high microwave field intensities to be achieved at much lower incident microwave power levels, thus greatly enhancing the signal-to-noise ratio in U'1 experiments. This reduction in the noise level made it possible to benefit from the other advantages of U'1 over V'2, such as stronger signals, simpler line shapes, and simpler data analysis. For these muscle fiber samples, the resulting sensitivity (signal/noise/sample volume) of the U'1 signals was greater than 100 times that of V'2 signals obtained in a conventional cavity. Another advantage of the U'1 display is that signals from weakly immobilized probes, i.e., probes that have nanosecond rotational mobility relative to the labeled protein (myosin), are greatly suppressed relative to strongly immobilized probes. This reduces the ambiguity of spectral analysis, and eliminates the need for chemical treatments [e.g., using K3Fe(CN)6] that were previously required in muscle fibers and other systems. Further suppression of this weakly immobilized component was achieved in U'1 spectra by increasing the microwave power and decreasing the field modulation frequency.

Actomyosin↗
PubMedDate not supplied

[Effect of temperature and degree of hydration on the mobility of spin labels in surface layers of proteins].

The mobility of separate sites of the water-protein matrix depending on temperature and degree of hydration has been investigated by means of spin labels covalently attached to surface layers of proteins (alpha-chymotrypsin and human serum albumin) and also by a spin probe in a hydrophobic "pocket" of human serum albumin. The results obtained are compared with the data on the mobility of gamma-resonance labels (57Fe) firmly bound with the protein matrix in the same samples. At certain temperature and degree of hydration both spin and gamma-resonance label show an increase in mobility. With the degree of hydration increasing one may observe a simultaneous increase in energy and in entropy of activation: rotatory diffusion of spin labels, i. e., a compensation effect takes place which confirms the concept expressed earlier that cooperation of water-protein interactions is the main reason of CEF. It should be noted that at P/PS greater than 0.8 the values of delta E =7 divided by 10 kcal/mole, and delta S not equal to = 9 divided by 11 e. e. are specific to glycerol-like systems, i. e., under these conditions (P/Ps greater than 0.8) the water-protein layer has glycerol-like properties.

Calorimetry↗
PubMed2006

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe.

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70 A in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40 A, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA-protein complexes.

DNA↗