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At least 703 records · Page 39Linked to original sources
PubMed2004

Solvent interactions and protein dynamics in spin-labeled T4 lysozyme.

Aspects of T4 lysozyme dynamics and solvent interaction are investigated using atomically detailed Molecular Dynamics (MD) simulations. Two spin-labeled mutants of T4 lysozyme are analyzed (T4L-N40C and T4L-K48C), which have been found from electronic paramagnetic resonance (EPR) experiments to exhibit different mobilities at the site of spin probe attachment (N- and C-terminus of helix B, respectively). Similarities and differences in solvent distribution and diffusion around the spin label, as well as around exposed and buried residues within the protein, are discussed. The purpose is to capture possible strong interactions between the spin label (ring) and solvent molecules, which may affect EPR lineshapes. The effect of backbone motions on the water density profiles is also investigated. The focus is on the domain closure associated with the T4 lysozyme hinge-bending motion, which is analyzed by Essential Dynamics (ED). The N-terminus of helix B is found to be a "hinge" residue, which explains the high degree of flexibility and motional freedom at this site.

Bacteriophage T4↗
PubMed1975

ESR spectral analysis of the molecular motion of spin labels in lipid bilayers and membranes based on a model in terms of two angular motional parameters and rotational correlation times.

Electron spin resonance (ESR) spectral line shapes are calculated for a nitroxide spin-labeled molecule undergoing rapid restricted rotations (twisting) about its long molecular axis while simultaneously tumbling within a cone. Explicit expressions are derived for the hyperfine splittings and g-values, as well as for the secular contributions to the motionally modulated linewidths. The present model is useful for analyzing the restricted twisting and tumbling motions, and rotational correlation times, of spin-labeled molecules in bilayers. Simulated spectra compare well with experimental spectra of lecithin bilayers marked with cholestane spin label, over a wide temperature range.

Cholestanes↗
PubMed1978

A photochemical technique for measuring lateral diffusion of spin-labeled phospholipids in membranes.

A technique is described for the measurement of lateral diffusion of any spin-labeled molecule in planar phospholipid multilayers, using low probe concentrations and a photochemical reaction to generate the initial concentration gradient. Features of the technique include simplicity of data analysis, versatility, and applicability over a wide range of motional rates. Measurements have been made on dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine in the fluid phase (using a phospholipid with head-group spin label), and excellent agreement is found with recent nuclear magnetic resonance spin-echo measurements, as well as with other spin-label and fluorescence photobleaching results.

Journal Article↗
PubMed1982

Binding and incorporation of lecithin-cholesterol vesicles to lymphocytes: a spin-label study.

When lecithin-cholesterol vesicles, containing the membrane-bound spin probe 3-doxyl-cholestane, were set in contact with mouse lymphocytes, the vesicles adsorbed to the cell and vesicle-membrane components were transferred to it. The spin probe was enzymatically reduced at the inside of the cell membrane. The spin-label method provided a means to determine quantitatively the extent of vesicles adsorption and vesicle-cell fusion by measuring the transfer of vesicles membrane material to the cell. This method, together with the reduction of spin label by the cell, allowed also a quantitative estimate to the extent of endocytosis during cell-liposome interaction.

Animals↗
PubMed1989

Synthesis of spin-labeled 2-azido-ATP: evidence for distinct nucleotide-binding sites in calcium pump protein from sarcoplasmic reticulum.

A spin-labeled and photoreactive derivative of ATP was synthesized with the spin label attached to the 2'- or 3'-position of the ribose moiety and an azido group to C2 of the adenine ring (SL-2N3-ATP). Irradiation of this compound at 350 nm generates a nitrene, which then reacts with nucleophiles in its vicinty. SL-2N3-ATP, in the presence of Ca2+, was hydrolyzed by the calcium pump protein (Ca2+-ATPase) of fast twitch skeletal muscle sarcoplasmic reticulum. The SL-2N3-ATP-enzyme complex in the absence of Ca2+ exhibited strongly immobilized ESR spectra. ESR spectra obtained after covalent incorporation of SL-2N3-ATP into Ca2+-ATPase and removal of freely tumbling SL-2N3-ATP exhibited motionally constrained species indicative of distinct and possibly adjacent ATP-binding sites. By contrast, with SL-ATP devoid of the azido group or with the corresponding 'non-cleavable' beta, gamma-methylene triphosphate analogue (SL-AMP-PCP), two distinct sites were not as well resolved in the ESR spectra due to spectral overlap with the signal from the freely tumbling fraction even with the enhanced spectral resolution provided by perdeuteration of the spin label. Thus, SL-2N3-ATP may have general application for ESR studies of ATP-dependent proteins under conditions in which non-covalent interactions are too weak for motionally restricted species to be resolved.

Adenosine Triphosphate↗
PubMed1977

Immune lysis of spin label loaded liposomes incorporating cardiolipin; a new sensitive method for detecting anticardiolipin antibodies in syphilis serology.

Liposomes prepared from a mixture of the pure lipids cholesterol, lecithin and cardiolipin (molar ratio 50/45/5), are able to bind antibodies directed against Treponema pallidum. When the liposomes are loaded with the water soluble spin label tempocholine chloride, the release of spin label from the liposomes can be monitored directly by observing changes in the paramagnetic resonance (ESR) spectrum from the spin label. The method offers a convenient technique for monitoring the complement-mediated lysis of liposomes, and may be applied in the serological diagnosis of syphilis, and a method for quantitative measurement of complement.

Animals↗
PubMed2003

Substrate-induced conformational changes of the periplasmic N-terminus of an outer-membrane transporter by site-directed spin labeling.

The structure and dynamics of the N-terminal and core regions of BtuB, an outer membrane vitamin B(12) transporter from Escherichia coli, were investigated by site-directed spin labeling. Cysteine mutants were generated by site-directed mutagenesis to place spin labels in the N-terminal region (residues 1-17), the core region (residues 25-30), and double labels into the Ton box (residues 6-12). BtuB mutants were expressed, spin labeled, purified, and reconstituted into phosphatidylcholine. In the presence of substrate (vitamin B(12)), EPR spectroscopy demonstrates that there is a conformational change in the Ton box similar to that seen previously for BtuB in intact outer membranes. The Ton box is positioned within the beta-barrel of BtuB in the absence of substrate (docked configuration) but becomes unfolded and increases its aqueous exposure upon substrate binding (undocked configuration). This conformational change and the similarity in the EPR spectra between reconstituted and native membranes indicate that BtuB is correctly folded and functional in the reconstituted system. The protein segment on the N-terminal side of the Ton box is highly mobile, and it becomes more mobile in the presence of substrate. Side chains in the region C-terminal to the Ton box also show increases in mobility with substrate addition, but position 16 appears to define a hinge point for this conformation change. EPR line shapes and relaxation data indicate that residues 25-30 form a beta-strand structure, which is analogous to the first beta-strand in the cores of the homologous iron transporters. When substrate binds to BtuB, this first beta-strand remains folded. The EPR spectra of double-nitroxide labels within the Ton box are broadened because of dipolar and collisional exchange interactions. The broadening pattern indicates that the Ton box is not helical but is in an extended or beta-strand structure.

Bacterial Outer Membrane Proteins↗
PubMed1986

Diradical nitroxyl spin label contrast agents for magnetic resonance imaging. A comparison of relaxation effectiveness.

The proton relaxation enhancement characteristics of seven potential MRI contrast agents containing two nitroxyl spin labels per molecule (diradicals) were compared with eight similar agents with only one spin label per molecule (monoradicals). Diradical nitroxyls were evaluated to test the hypothesis that multiple paramagnetic centers in one molecule will result in stronger proton relaxation enhancement characteristics, allowing effective contrast enhancement at lower molar concentrations and thus a reduced osmotic load and greater safety. The acute toxicity of these agents is believed to be largely related to osmotic load. Five of seven diradical nitroxyls tested had spin-lattice relaxivities that were substantially greater than all eight of the monoradicals tested. The spin-spin relaxation properties of these agents and other pertinent characteristics are favorable for contrast enhancement. The results indicate that diradical nitroxyl spin labels may be used advantageously for the design of safer, more effective MRI contrast agents.

Chemical Phenomena↗
PubMed1986

Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins.

New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.

Chromatography, Gel↗
PubMed1975

N-terminal spin label studies of hemoglobin, Ligand and pH dependence.

Human hemoglobin was spin labeled with 4-isothiocanato-2,2,6,6-tetramethyl-piperdinooxyl, which is known to bind specifically to the N-terminal alpha-amino groups of proteins and slightly to the reactive sulfhydryl groups. Electron spin resonance (ESR) analysis indicated a partially resolved five-line spectrum, suggesting that the label was attached to at least two different binding sites. Using specific blocking reagents prior to spin labeling, the two binding sites were attributed to the sulfhydryl group of beta-93 (immobile) and the alpha-amino group of the N-terminal valines (mobile). The relative motion of the spin at one set of binding sites was restricted regardless of the state of ligation and pH, while the motion at the other site showed dependence on those parameters, e.g. the spin-labeled N-terminal ends of deoxyhemoglobin have restricted motion at all pH ranges studied, while those of oxyhemoglobin are relatively free to move at the basic pH range, but become more restricted in the acidic pH range.

Electron Spin Resonance Spectroscopy↗
PubMed1989

Subcellular distribution of a nitroxide spin-labeled netropsin in living KB cells. Electron paramagnetic resonance and sequence specificity studies.

A nitroxide spin-labeled netropsin was studied by EPR spectroscopy with respect to its uptake and localization in living KB cells. Whereas the drug was taken up readily, there was relatively little drug in the cytoplasm, but a significant concentration of the drug in the cell nucleus. The EPR signal in the latter site corresponded to a relatively freely rotating radical. The drug exhibited good intracellular stability up to 25 hr. While a delta Tm of 24 degrees between the spin-labeled netropsin and calf thymus DNA confirmed strong binding, the absence of any DNA elongation by viscometry was consistent with nonintercalative exterior binding which was confirmed to be minor groove specific by binding of the agent to T4 DNA with a delta Tm of 17.5 degrees. The sequence specificity of the DNA binding of the spin-labeled drug was confirmed by methidiumpropyl-EDTA (MPE) footprinting on a fragment of pBR322 DNA to be very similar to that of the parent netropsin, i.e. selective for AT-rich sites, with minor differences of protection afforded by introduction of the nitroxide label.

Base Sequence↗
PubMed1984

Vesicular stomatitis virus induced membrane changes: a spin label study.

Synchronized entry of Vesicular Stomatitis Virus (VSV) into spin labeled cultured human cells resulted in an increase in the rigidity of cell membranes as measured by Electron Spin Resonance Spectroscopy. Treatment of spin labeled cells with homologous interferon alpha did not influence the membrane fluidity, neither did it significantly prevent the VSV induced membrane changes despite its anti-viral protection.

Carcinoma, Hepatocellular↗
PubMed2000

Nonlinear electron paramagnetic resonance studies of the interaction of cytochrome c oxidase with spin-labeled lipids in gel-phase membranes.

The interaction of lipids, spin-labeled at different positions in the sn-2 chain, with cytochrome c oxidase reconstituted in gel-phase membranes of dimyristoylphosphatidylglycerol has been studied by electron paramagnetic resonance (EPR) spectroscopy. Nonlinear EPR methods, both saturation transfer EPR and progressive saturation EPR, were used. Interaction with the protein largely removes the flexibility gradient of the lipid chains in gel-phase membranes. The rotational mobility of the chain segments is reduced, relative to that for gel-phase lipids, by the intramembranous interaction with cytochrome c oxidase. This holds for all positions of chain labeling, but the relative effect is greater for chain segments closer to the terminal methyl ends. Modification of the paramagnetic metal-ion centers in the protein by binding azide has a pronounced effect on the spin-lattice relaxation of the lipid spin labels. This demonstrates that the centers modified are sufficiently close to the first-shell lipids to give appreciable dipolar interactions and that their vertical location in the membrane is closer to the 5-position than to the 14-position of the lipid chains.

Animals↗
PubMed1997

Spatiotemporal ESR-CT study on the metabolism of spin-labeled polysaccharide in a mouse.

A spatiotemporal ESR-CT study, rapid three dimensional ESR imaging by which distribution and metabolism of radicals in a small region in a living body can be followed, was carried out by intravenously administering spin-labeled polysaccharides to mice. The in vivo lifetime of spin-labeled hydroxyethylstarch (TEMPO-HES) was shorter than that of spin-labeled dextran (TEMPO-DX), suggesting that the clearance of their spins depended on the stability of the polymer chains to the hydrolysis. Spatiotemporal image at the kidney domain of a mouse showed that low molecular weight TEMPO-DX was excreted into the kidney and then was transferred to the bladder. Time dependence of the ESR intensity of TEMPO-HES at certain points in the liver domain had a maximum showing that TEMPO-HES was taken into the liver and then was decomposed there by the metabolism, but the rate of decrease in the intensity was different in each part in the liver because of the difference in metabolic function. The rate of decrease of TEMPO-DX in the blood was slowed by the prior administration of dextran, meaning that the intake of TEMPO-DX into the liver from the blood was suppressed by the dextran.

Animals↗
PubMed2004

Membrane binding, structure, and localization of cecropin-mellitin hybrid peptides: a site-directed spin-labeling study.

The interaction of antimicrobial peptides with membranes is a key factor in determining their biological activity. In this study we have synthesized a series of minimized cecropin-mellitin hybrid peptides each containing a single cysteine residue, modified the cysteine with the sulfhydryl-specific methanethiosulfonate spin-label, and used electron paramagnetic resonance spectroscopy to measure membrane-binding affinities and determine the orientation and localization of peptides bound to membranes that mimic the bacterial cytoplasmic membrane. All of the peptides were unstructured in aqueous solution but underwent a significant conformational change upon membrane binding that diminished the rotational mobility of the attached spin-label. Apparent partition coefficients were similar for five of the six constructs examined, indicating that location of the spin-label had little effect on peptide binding as long as the attachment site was in the relatively hydrophobic C-terminal domain. Depth measurements based on accessibility of the spin-labeled sites to oxygen and nickel ethylenediaminediacetate indicated that at high lipid/peptide ratios these peptides form a single alpha-helix, with the helical axis aligned parallel to the bilayer surface and immersed approximately 5 A below the membrane-aqueous interface. Such a localization would provide exposure of charged/polar residues on the hydrophilic face of the amphipathic helix to the aqueous phase, and allow the nonpolar residues along the opposite face of the helix to remain immersed in the hydrophobic phase of the bilayer. These results are discussed with respect to the mechanism of membrane disruption by antimicrobial peptides.

Antimicrobial Cationic Peptides↗
PubMed1988

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes.

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1991

Activity and conformational changes of alpha-chymotrypsin in reverse micelles studied by spin labeling.

alpha-Chymotrypsin (CT), spin-labeled at the active site by using an acylating label which constitutes a substrate for this protein, has been investigated in reverse micelles formed by AOT in isooctane. The electron spin resonance spectra provided information on conformation, dynamics and deacylation activity. The dynamics of the label bound to CT appears to be more hindered in reverse micelles than in aqueous solution, probably owing to the effect of the micellar environment on protein conformation. The deacylation rate in reverse micelles does not show the characteristic bell-shaped dependence on water content which is generally found for CT enzymatic activity.

Acylation↗
PubMed1994

Superoxide scavenging activity of spin-labeled nitrosourea and triazene derivatives.

Superoxide scavenging activities (SSA) of newly synthesized spin-labeled nitrosourea and triazene derivatives, and their precursor nitroxides were investigated by the ESR/spin-trapping method using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and hypoxanthine/xanthine oxidase as the superoxide-generating system. The spin-labeled nitrosoureas, triazenes and their precursor nitroxides exhibited excellent SSA, whereas clinically used nitrosourea and triazene, which do not contain the nitroxide moiety, did not show any SSA. Furthermore, it was deduced that these nitroxides scavenge superoxide by redox cycling between nitroxide and corresponding hydroxylamine.

Electron Spin Resonance Spectroscopy↗