Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Spin Labels”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,333 records · Page 74Linked to original sources
PubMed1998

Selectivity of lipid-protein interactions with trypsinized Na, K-ATPase studied by spin-label EPR.

The selectivity of the lipid-protein interactions in trypsinised Na, K-ATPase membranes from Squalus acanthias has been determined by using EPR spectroscopy with different lipid probes spin-labelled on the 14-C atom of the fatty acid chain. From measurements at low ionic strength and different pH values, the pattern of selectivity is: (stearic acid)->(phosphatidylserine)->(stearic acid)0>(phosphatidylcholine)+/-, where superscripts indicate the formal electrostatic charge on the lipid headgroup. This is in the same order as that determined with native Na,K-ATPase membranes [M. Esmann, D. Marsh, Biochemistry 24 (1985) 3572-3578]. The selectivity for phosphatidylserine is independent of pH, over the range pH 6.0-9. 0, as found also for native membranes. For membranes trypsinised in the presence of Rb+ ions, and in the presence of Na+ (which allows more extensive proteolysis), the relative association constants, Kr, of all lipids are the same as for control membranes, with the exception of ionised (stearic acid)- that shows the highest specificity. Therefore, both the stoichiometry and the principal determinants of the specificity of lipid-protein interaction are preserved on extensive trypsinisation of Na,K-ATPase membranes. This has implications for the location and arrangement of those amino acid side chains that determine the lipid selectivity of the native Na,K-ATPase.

Animals↗
PubMed2006

Distance measurements by fluorescence energy homotransfer: evaluation in T4 lysozyme and correlation with dipolar coupling between spin labels.

We demonstrate the feasibility and practical limitations of using steady-state anisotropy to determine distances from fluorescence homotransfer in the context of a protein of known crystal structure. Eight double mutants of T4 lysozyme spanning the distance range between 20 A and 50 A were labeled with a methanethiosulfonate derivative of fluorescein. The measured distances in liquid solution are in agreement with those determined from dipolar coupling between spin labels in the frozen state. They can be interpreted in the context of the crystal structure after accounting for the probe linking arm. Overall, the results establish the necessary calibration for this spectroscopic ruler. The measurement of similar distance trends using independent probes sets the stage for the complementary use of homotransfer and dipolar coupling in the determination of static structures and detection of conformational changes.

Anisotropy↗
PubMed1993

Transient detection of spin-labeled myosin subfragment 1 conformational states during ATP hydrolysis.

We have used time-resolved electron paramagnetic resonance spectroscopy and caged ATP to detect nucleotide-induced changes in the conformational state of spin-labeled myosin heads (IASL-S1). Changes in the internal rotational dynamics of IASL-S1 were monitored with millisecond time resolution during the pre-steady-state phase of ATP hydrolysis. The changes in the internal protein dynamics were rigorously correlated with specific biochemical kinetic transitions, allowing us to observe directly the dynamic sequence of structural changes in IASL-S1 during the binding and hydrolysis of ATP. When caged ATP was photolyzed (producing 500 microM ATP) in the presence of 100 microM IASL-S1, the EPR signal intensity rose transiently to the steady-state ATPase level, indicating increased rotational motion about the SH1 region of the myosin head. Kinetic and spectral analyses have resolved two phases of this transient, one representing the population of the M*.ATP state and the other representing the population of the M**.ADP.Pi state. We conclude that two motionally distinct states of the myosin head are present during ATP hydrolysis and that these states represent distinct conformational states that can be correlated with specific biochemical intermediates. Since specific labeling of myosin heads with IASL has been achieved in skinned muscle fibers, this study establishes the feasibility for the first direct detection and resolution of myosin's conformational transients during muscle contraction.

Adenosine Diphosphate↗
PubMed1994

Tissue distribution of polybutylcyanoacrylate nanoparticles loaded with spin-labelled nitrosourea in Lewis lung carcinoma-bearing mice.

The tissue distribution of polybutylcyanoacrylate nanoparticles (PBCN) with a diameter of 127 nm, loaded with 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6-tetramethylpiperidinyl)-1- nitrosourea (spin-labelled nitrosourea, SLCNU) is described. PBCN-suspensions were intraperitoneally (i.p.) injected into Lewis lung carcinoma bearing mice. The biodistribution of PBCN in the visceral organs, blood and tumor was studied by electron spin resonance (ESR) spectroscopy. A relatively low accumulation of nanoparticles in the liver and spleen was found. The accumulation was negligible in the i.m. implanted primary tumor. SLCNU-loaded nanoparticles were mainly found in the lungs, kidneys, and heart. The highest content of the particles studied was observed in the lungs of tumor bearing experimental animals damaged by metastases. These findings suggest that PBCN offer some opportunities in the targeting of SLCNU to lung metastases.

Animals↗
PubMed1999

Site-directed spin labeling study of subunit interactions in the alpha-crystallin domain of small heat-shock proteins. Comparison of the oligomer symmetry in alphaA-crystallin, HSP 27, and HSP 16.3.

Site-directed spin labeling was used to investigate quaternary interactions along a conserved sequence in the alpha-crystallin domain of alphaA-crystallin, heat-shock protein 27 (HSP 27), and Mycobacterium tuberculosis heat-shock protein (HSP 16.3). In previous work, it was demonstrated that this sequence in alphaA-crystallin and HSP 27 forms a beta-strand involved in subunit contacts. In this study, the symmetry and geometry of the resulting interface were investigated. For this purpose, the pattern of spin-spin interactions was analyzed, and the number of interacting spins was determined in alphaA-crystallin and HSP 27. The results reveal a 2-fold symmetric interface consisting of two beta-strands interacting near their N termini in an antiparallel fashion. Remarkably, subunit interactions along this interface persist when the alpha-crystallin domains are expressed in isolation. Because this domain in alphaA-crystallin forms dimers and tetramers, it is inferred that interactions along this interface mediate the formation of a basic dimeric unit. In contrast, in HSP 16.3, spin-spin interactions are observed at only one site near the C terminus of the sequence. Furthermore, cysteine substitutions at residues flanking the N terminus resulted in the dissociation of the oligomeric structure. Analysis of the spin-spin interactions and size exclusion chromatography indicates a 3-fold symmetric interface. Taken together, our results demonstrate that subunit interactions in the alpha-crystallin domain of mammalian small heat-shock proteins assemble a basic building block of the oligomeric structure. Sequence divergence in this domain results in variations in the size and symmetry of the quaternary structure between distant members of the small heat-shock protein family.

Amino Acid Sequence↗
PubMed1985

Effect of the presence of hardened erythrocytes on deformation-orientation characteristics of normal erythrocytes in shear flow studied by the spin label method.

The effect of the presence of hardened red blood cells (HRBC) in a mixed suspension on the deformation-orientation characteristics of normal cells in flow is experimentally probed by the electron spin resonance (ESR) spin label method, using a phosphatidylcholine label which does not transfer between the cells. The average deformation-orientation of the normal cells is generally suppressed by the presence of HRBC to different degrees, depending upon the shape and the way the HRBC are prepared. The effects are qualitatively explained by disturbance of laminar shear flow due to the random tumbling of the HRBC.

Blood Flow Velocity↗
PubMed2005

Force field impact and spin-probe modeling in molecular dynamics simulations of spin-labeled T4 lysozyme.

Several attempts have been made to compute electron paramagnetic resonance (EPR) spectra of biomolecules, using motional models or simulated trajectories to describe dynamics. Ideally, the simulated trajectories should capture "fast" (picosecond) snapshots of spin-probe rotations accurately, while being lengthy enough to ensure a proper Fourier integration of the time-domain signal. It is the interplay of the two criteria that poses computational challenges to the method. In this context, an analysis of the spin-probe and protein conformational sampling and equilibration, with different force fields and with explicit solvent, may be a useful attempt. The present work reports a comparative study of the effect of the molecular dynamics (MD) force field on conformational sampling and equilibration in two spin-labeled T4 lysozyme (T4L) variants, N40C and K48C. Ensembles of 10x 3 ns-trajectories per variant and per force field (OPLS/AMBER and AMBER99) are analyzed for a reliable assessment of convergence and sampling. It is found that subtle site-dependent differences in spin-probe rotations and torsions are more readily captured in the AMBER99 trajectories than in the OPLS/AMBER simulations. On the other hand, sampling and equilibration are found to be better with the OPLS/AMBER force field at equal trajectory lengths.

Bacteriophage T4↗
PubMed2005

Dipolar coupling between nitroxide spin labels: the development and application of a tether-in-a-cone model.

A tether-in-a-cone model is developed for the simulation of electron paramagnetic resonance spectra of dipolar coupled nitroxide spin labels attached to tethers statically disordered within cones of variable halfwidth. In this model, the nitroxides adopt a range of interprobe distances and orientations. The aim is to develop tools for determining both the distance distribution and the relative orientation of the labels from experimental spectra. Simulations demonstrate the sensitivity of electron paramagnetic resonance spectra to the orientation of the cones as a function of cone halfwidth and other parameters. For small cone halfwidths (< approximately 40 degrees ), simulated spectra are strongly dependent on the relative orientation of the cones. For larger cone halfwidths, spectra become independent of cone orientation. Tether-in-a-cone model simulations are analyzed using a convolution approach based on Fourier transforms. Spectra obtained by the Fourier convolution method more closely fit the tether-in-a-cone simulations as the halfwidth of the cone increases. The Fourier convolution method gives a reasonable estimate of the correct average distance, though the distance distribution obtained can be significantly distorted. Finally, the tether-in-a-cone model is successfully used to analyze experimental spectra from T4 lysozyme. These results demonstrate the utility of the model and highlight directions for further development.

Anisotropy↗
PubMed1997

Structure and function of the conserved domain in alphaA-crystallin. Site-directed spin labeling identifies a beta-strand located near a subunit interface.

Twelve sequential single cysteine mutants of alphaA-crystallin extending between amino acids Y109 and L120 were prepared and reacted with a sulfhydryl specific spin label in order to investigate the role of this sequence in the assembly of the alphaA-crystallin quaternary structure and its chaperone-like function. The sequence is located in the region of highest homology in the alpha-crystallin domain, a stretch of 100 amino acids conserved among lens alpha-crystallins and small heat-shock proteins (sHSPs). Analysis of the solvent accessibility and mobility of the attached nitroxides reveals that the sequence, as a whole, is relatively sequestered from the aqueous solvent. Furthermore, as teh nitroxide is scanned across the sequence, both mobility and accessibility vary with a periodicity of 2, demonstrating that the backbone conformation is that of a beta-strand. Once face of the strand, containing the highly conserved residues R112 and R116, is buried with virtually no accessibility to the aqueous solvent. Equivalent strands from different subunits are in close spatial proximity, as inferred from spin-spin interactions between identical residues along the strand. Taken together, our results are consistent with the hypothesis that the alpha-crystallin domain is a building block of the alpha-crystallins quaternary structure and suggest that the charge conservation observed in the alpha-crystallins evolution might be important for the assembly of the oligomer. This work reports the first use of SDSL to identify a beta-strand in an unknown structure and demonstrates the feasibility of using this technique to investigate the oligomeric structure of the alpha-crystallins and sHSPs.

Crystallins↗
PubMed1982

Equilibrium binding of spin-labeled fatty acids to bovine serum albumin: suitability as surrogate ligands for natural fatty acids.

Electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies were used to characterize the binding of spin-labeled fatty acid (SLFA) to bovine serum albumin (BSA). Association constants of three stearic acid derivatives labeled with a nitroxyl radical at C-5, C-12, or C-16 were estimated by EPR spectroscopy as the ratio of SLFA to BSA was increased from about 0 to 9. The values were compared to those for unmodified stearate. With all three SLFA, it was apparent that the nitroxyl residue modified the binding pattern. For SLFA:BSA ratios up to 1, which probably involves the site(s) on BSA most specific for long-chain FA, the C-16 derivative bound with an affinity similar to that of the natural FA. At higher ratios, the association constants for this SLFA were lower than those for stearate. The C-12 and C-5 derivatives showed only low-affinity binding relative to stearate. The spectral parameter, W, was constant for SLFA:BSA ratios between 0 and 1 in the case of C-16 compound, indicating physical homogeneity of the high-affinity binding site. At higher ratios, the spectra changed progressively, indicating inhomogeneity of the lower affinity binding sites although parallel changes in association constants were not observed. Changes in W due to Heisenberg spin exchange were ruled out. By examining the mobility profile of the bound SLFA by both EPR and ST-EPR techniques, it was shown that the nitroxyl group was maximally immobilized when attached near the center of the carbon chain of the bound SLFA.

Binding, Competitive↗
PubMed2001

Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling.

SNARE (soluble NSF acceptor protein receptor) proteins are thought to mediate membrane fusion by assembling into heterooligomeric complexes that connect the fusing membranes and initiate the fusion reaction. Here we used site-directed spin labeling to map conformational changes that occur upon homo- and heterooligomeric complex formation of neuronal SNARE proteins. We found that the soluble domains of synaptobrevin, SNAP-25, and syntaxin 1 are unstructured. At higher concentrations, the SNARE motif of syntaxin 1 forms homooligomeric helical bundles with at least some of the alpha-helices aligned in parallel. In the assembled SNARE complex, mapping of thirty side chain positions yielded spectra which are in good agreement with the recently published crystal structure. The loop region of SNAP-25 that connects the two SNARE motifs is largely unstructured. C-terminal truncation of synaptobrevin resulted in complexes that are completely folded N-terminal of the truncation but become unstructured at the C-terminal end. The binary complex of syntaxin and SNAP-25 consists of a parallel four helix-bundle with properties resembling that of the ternary complex.

Amino Acid Substitution↗
PubMed2005

Continuous arterial spin labeling using a train of adiabatic inversion pulses.

PURPOSE: To develop a simple and robust magnetic resonance imaging (MRI) pulse sequence for the quantitative measurement of blood flow in the brain and cerebral tumors that has practical implementation advantages over currently used continuous arterial spin labeling (CASL) schemes. MATERIALS AND METHODS: Presented here is a single-coil protocol that uses a train of hyperbolic secant inversion pulses to produce continuous arterial spin inversion for perfusion weighting of fast spin echo images. Flow maps of normal rat brains and those containing a 9L gliosarcoma orthotopic tumor model conditions were acquired with and without carbogen. RESULTS: The perfusion-weighted images have reduced magnetization transfer signal degradation as compared to the traditional single-coil CASL while avoiding the use of a more complex two-coil CASL technique. Blood flow measurements in tumor and normal brain tissue were consistent with those previously reported by other CASL techniques. Contralateral and normal brain showed increased blood flow with carbogen breathing, while tumor tissue lacked the same CO(2) reactivity. CONCLUSION: This variation of the CASL technique is a quantitative, robust, and practical single-coil method for measuring blood flow. This CASL method does not require specialized radiofrequency coils or amplifiers that are not routinely used for anatomic imaging of the brain, therefore allowing these flow measurements to be easily incorporated into traditional rodent neuroimaging protocols.

Anesthetics, Inhalation↗
PubMed1997

Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. Paramagnetic relaxation enhancement by nitroxide spin labels.

Structural analysis of delta131delta, a fragment model of the denatured state of staphylococcal nuclease, has been extended by obtaining long-range distance restraints between protein chain segments based on paramagnetic relaxation enhancement methods. PROXYL spin labels were attached at unique cysteine residues introduced at 14 different sites along the polypeptide chain, and the resulting enhancements of amide proton relaxation were measured by NMR spectroscopy. To minimize perturbation of denatured state structure, these labeling sites were chosen on the basis of a high solvent exposure in the native state and a small change in stability and m-value upon mutation of the wild-type residue to cysteine or alanine. EPR spectroscopy confirmed that in all cases the PROXYL label of the modified protein was solvent-exposed and undergoing free isotropic rotation. By quantifying at 500 MHz and 600 MHz the enhancement of both T1 and T2 relaxation for amide protons resolved in a 1H-15N correlation spectrum, the apparent correlation time for the free electron-proton vectors for six PROXYL-labeled proteins could be estimated. With these data plus the enhancements in transverse relaxation rate (R2) for the other eight proteins, the time-averaged, r(-6) weighted distance between the free electron on the unique nitroxide and 30 to 60 amide protons in each protein could be approximated. Inspection of the pattern of R2 enhancements reveals a significant amount of long-range structure in this denatured state, a clear indication that it is not a random coil.

Amides↗
PubMedDate not supplied

[Study of lysozyme by a spin-label method in the 2-mm range].

Two millimeter range ESR-spectroscopy was used to measure the values of magnetic resonance parameters of egg lysozyme samples modified with nitroxyl label 4-(iodine-acetamide)-2,2,6,6-tetramethylpiperidine-1-oxyl by hist-15 residue. It has been shown that in a lyophilic sample the spin label forms a hydrogen bond with the protein group and when the sample is moistened--with water molecules. Temperature changes of the pattern of ESR line are analysed. It is concluded that in the moistened samples within 230-320 K the nitroxyl fragment of the label gets engaged in anisotropic movement with preferable rotation around Z-axis of N-O. fragment with anisotropy coefficient 5 and mean correlation time tau congruent to 5 X 10(-7) divided by 5 X 10(-8) c.

Electron Spin Resonance Spectroscopy↗
PubMed1993

Using nitroxide spin labels. How to obtain T1e from continuous wave electron paramagnetic resonance spectra at all rotational rates.

Historically, the continuous wave electron paramagnetic resonance (CW-EPR) progressive saturation method has been used to obtain information on the spin-lattice relaxation time (T1e) and those processes, such as motion and spin exchange, that occur on a competitive timescale. For example, qualitative information on local dynamics and solvent accessibility of proteins and nucleic acids has been obtained by this method. However, making quantitative estimates of T1e from CW-EPR spectra have been frustrated by a lack of understanding of the role of T1e (and T2e) in the slow-motion regime. Theoretical simulation of the CW-EPR lineshapes in the slow-motion region under increasing power levels has been used in this work to test whether the saturation technique can produce quantitative estimates of the spin-lattice relaxation rates. A method is presented by which the correct T1e may be extracted from an analysis of the power-saturation rollover curve, regardless of the amount of inhomogeneous broadening or the rates of molecular reorientation. The range of motional correlation times from 10 to 200 ns should be optimal for extracting quantitative estimates of T1e values in spin-labeled biomolecules. The progressive-saturation rollover curve method should find wide application in those areas of biophysics where information on molecular interactions and solvent exposure as well as molecular reorientation rates are desired.

Biophysical Phenomena↗
PubMed1994

Molecular organization and dynamics in bacteriorhodopsin-rich reconstituted membranes: discrimination of lipid environments by the oxygen transport parameter using a pulse ESR spin-labeling technique.

Molecular organization and dynamics in protein-rich membranes have been studied by investigating transport (diffusion-concentration product) of molecular oxygen at various locations in reconstituted membranes of bacteriorhodopsin (BR) and L-alpha-dimyristoylphosphatidylcholine. Oxygen transport was evaluated by monitoring the bimolecular collision of molecular oxygen with four types of nitroxide lipid spin labels placed at various locations in the membrane. The collision rate was estimated from the spin-lattice relaxation times (T1's) measured at various oxygen partial pressures by analyzing the short-pulse saturation recovery ESR signals. CD spectra and decay of polarized flash-induced photodichroism of bacteriorhodopsin indicated that BR molecules are monomers in reconstituted membranes with a lipid/BR molar ratio of 80 (80-rec) and are 25% monomers and 75% trimers plus oligomers of trimers when the lipid/BR ratio is 40 (40-rec). In the 80-rec, the lipid environment is homogeneous on a microsecond scale (T1), probably because the exchange rate of lipids between the bulk and the boundary regions is greater than the T1 relaxation rate (approximately 10(6) s-1). The oxygen collision rate in the hydrophobic region of the 80-rec membrane is smaller by a factor of 1.6 than in that of the lipid membrane without BR, and the effect of BR in decreasing the collision rate is independent of the "depth" in the hydrophobic region. In the 40-rec, two collision rates were observed, one of which is close to those for purple membrane (or the gel-phase membrane), while the other is about the same as was measured in the 80-rec.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed2001

Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N3-SL-ATP.

2-N3-SL-ATP [2-azido-2',3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl-pyrroline) adenosine triphosphate], a photoaffinity spin-labeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotide-binding site stoichiometry of sarcoplasmic reticulum (SR) Ca2+-ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spin-spin interactions between two radicals corresponding to a distance of about 15 A between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca2+-ATPase is a dimer with the nucleotide-binding sites in close proximity.

Adenosine Triphosphate↗