Electron spin resonance study of the synaptosome opiate receptor: kinetics of stereospecific binding of spin labeled morphine.

Morphine, spin labeled on the 3- or 6-position has been used as the opiate ligand in a study of the time course of stereospecific opiate binding to intact synaptosomes isolated from non-cerebellar rat brain. The broadening of electron spin resonance lines induced by immobilization of the ligand on binding has been used to determine the concentration of bound opiate. The stereospecificity of the reaction was measured by comparing ligand binding in the presence of thousand-fold molar excesses of dextrorphan or levorphanol. Using both static and flow techniques, the binding process has been continuously monitored at times greater than 4.8 s after mixing spin labeled morphine with synaptosomes. It is shown that for this ligand and receptor preparation, binding takes place primarily during a delayed, abrupt process whose rate and time of onset are temperature dependent and reflect the presence of added opiate agonist or antagonist.     

Kinetics of inhibition of acetylcholinesterase by spin labeled acetylcholine analogs.

A series of spin labeled acetycholine analogs, in which the number of methylene groups between the quaternary nitrogen and the alcohol oxygen ranged between 1-5, have been examined as inhibitors of electric eel acetylcholinesterase. Evidence is presented suggesting that inhibition of acetylocholinesterase by the spin labeled ACH analogs is due to the high affinity of these compounds for the enzyme, inhibition is competitive and reversible. It has been shown that complex formation is of major importance in the reaction between spin labeled ACH analogs and acetylcholinesterase. The acetylation step has been shown to occur by demonstrating that the leaving group is released as the reaction proceeds. Complex formation has been demonstrated by means of kinetic criteria. Kinetic parameter have been measured for the five compounds, and correlations with alkaline hydrolysis are disussed.     

Rotational diffusion and intermolecular collisions of a spin labeled alpha-helical peptide determined by electron spin echo spectroscopy.

Short peptides that are composed mainly of alanine have recently been shown to form alpha-helices in aqueous solution at low temperature (Marqusee, S., and R. L. Baldwin. 1987. Proc. Natl. Acad. Sci. 84:8898-8902; Marqusee, S., V. H. Robbins, and R. L. Baldwin. 1989. Proc. Natl. Acad. Sci. USA. 86:5286-5290). These peptides are excellent models for probing structure and dynamics in isolated helical domains. In previous work we have designed and synthesized spin labeled analogs of these helix-forming peptides and we have shown that these analogs retain the folding characteristics of the parent peptide (Todd, A. P., and G. L. Millhauser. 1991. Biochemistry. 30:5515-5523). Using conventional continuous wave electron spin resonance (CW ESR) we have further shown that local motion is more pronounced near the helix amino terminus than in the central region as the peptide is thermally unfolded (Miick, S. M., A. P. Todd, and G. L. Millhauser. 1991. Biochemistry. 30:9498-9503). In this present work we use electron spin echo (ESE) spectroscopy to further refine our understanding of the solution dynamics of the 3K-8 peptide, which is a 16-mer with a nitroxide spin label attached at position 8. We find that the spin echo decays are well described by a single exponential function and that the determined correlation times are close to those previously derived from CW experiments. Variable concentration ESE experiments have directly revealed Heisenberg spin exchange (HSE) interactions and we find that the interpeptide collision rate is near to that expected for a free species in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Superoxide formation in spinach chloroplasts: electron spin resonance detection by spin trapping.

The new technique of spin trapping has been applied to a biological system for the first time. The light induced generation of O₂^? by chloroplasts in the presence of oxygen has been shown by the production of the O₂^? adduct of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide. The O₂^? adduct was detected by electron spin resonance spectroscopy. Methyl viologen enhanced the production of the O₂^? adduct thus providing support for the hypothes is that methyl viologen accepts electrons from the primary acceptor of photosystem I and subsequently reduces O₂ to O₂^?.     

Radical anions from one-electron-reduced adrenochrome. Detection and identification by electron spin resonance spectroscopy

Free radicals from the one-electron reduction of adrenochrome have been studied in aqueous solutions. These radicals have been detected and identified by electron spin resonance spectroscopy, using spin stabilization methods (complexation with diamagnetic metal ions) to enhance radical concentrations. It is shown that the radicals have a characteristic ESR spectrum enabling their identification in complex systems. The spin density distribution in the radicals has been studied as a function of complexing metal ions and solvent composition. In the presence of oxidants (e.g., oxygen) the spectrum of the radical is replaced by that derived from the one-electron exidation of adrenochrome.     

Light-dependent spin trapping of hydroxyl radical from human erythrocytes.

The generation of reactive oxygen species from human erythrocytes has previously been demonstrated. Furthermore, erythrocytic protoporphyrin IX has been shown to generate superoxide and singlet oxygen when exposed to light. These findings suggest that a component of erythrocytic reactive oxygen species production may be light-dependent. By inhibiting erythrocyte superoxide dismutase, catalase, and glutathione peroxidase with N,N-diethyldithiocarbamate or sodium cyanide, we demonstrate the light-dependent generation of hydroxyl radical in human erythrocytes using spin trapping/Electron Spin Resonance spectroscopy. This finding may be significant in tissues where blood is exposed to light, such as in the eye.     

Electron spin resonance study of human alpha 1-acid glycoprotein interaction with a spin labelled steroid.

The interaction of human alpha 1-acid glycoprotein (AAG) with a corticosteroid was studied using nitroxide labeled deoxycorticosterone and electron spin resonance (ESR) spectroscopy. The ESR spectra of the spin labeled steroid in the presence of AAG could be used to characterize the ligand-protein interaction at equilibrium without the need of a separation between bound and free species. An association constant Ka of 6.10(5) M-1 at 20 degrees C and a binding capacity of one site per mole protein were found. ESR spectra recorded at equilibrium at various temperatures allowed the calculation of enthalpy and entropy variations for the steroid-protein interaction; these thermodynamic parameters exhibited a rapid change above 45 degrees C which may be related to a protein conformational modification above this temperature, as detected by circular dichroism study. The ESR spectra width could be used to define a polar character for the spin label environment in the steroid binding site of AAG and to calculate an apparent rotational correlation time of 2.8 x 10(-8) sec for the steroid-protein complex in aqueous solution at 20 degrees C. It can be concluded that spin labeling and ESR methodology is of value in the study of steroid-protein interactions of biological significance above all because it can provide direct physico-chemical information concerning the local environment of the ligand in its binding site at equilibrium.     

Motional restrictions of membrane proteins: a site-directed spin labeling study

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.     

Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy

The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using a water-soluble spin relaxation agent, chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (<0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.     

Synthesis of some steroid spin labels

An improved synthesis of spin-labeled cortisol, cortisone, and testosterone using N,N'-cyclohexylcarbodiimide (DCC) is reported. The spin labeled steroids are shown to result from the esterification at the most reactive C(21) and C(17) hydroxyl groups. A mild and high yield oxidation of cortisol spin label to cortisone spin label by chromium trioxide-pyridine is also discussed.     

Saturation transfer electron paramagnetic resonance spectroscopy of spin labeled tobacco mosaic virus protein.

The coat protein of Tobacco Mosaic Virus is covalently labeled with a maleimide spin label at the single SH-group of the protein. Saturation transfer electron paramagnetic resonance spectroscopy, a technique that is sensitive to very slow molecular motion with rotational correlation times τ_c in the range 10^?7 to 10^?3 sec, shows the dissociation of large oligomers of spin labeled protein with τ_c～10^?4 sec at pH 5.5 to smaller oligomers at higher pH.     

Detection of drug‐induced conformational change of a transmembrane protein in lipid bilayers using site‐directed spin labeling

As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high‐resolution structural data available for the M2 protein to design and interpret site‐directed spin‐labeling electron paramagnetic resonance spectroscopy experiments on drug‐induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22–46 and M2TMC 23–60). M2TM peptides were spin labeled at the N‐terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C‐terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C‐terminal amphipathic helices.     

A ganglioside spin label: ganglioside head group interactions.

A general procedure has been developed for covalent attachment of a nitroxide spin label in the head group region of gangliosides. Gangliosides so labeled and incorporated into lipid bilayer vesicles give a sharp, 3-line spectrum characteristic of a highly mobile structure. The molecular basis of apparent ganglioside-ganglioside head group interaction is briefly discussed.     

Study of ubiquinone binding in ubiquinol-cytochrome c reductase by spin labeled ubiquinone derivative

A functionally active, spin labeled ubiquinone derivative, 2,3-dimethoxy -5-methyl-6-{10-(2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl-3-carboxy)-decyl}-1,4-benzoquinone, has been synthesized for the study of ubiquinone binding in ubiquinol-cytochrome $\text{c}$ reductase. When this spin labeled ubiquinone derivative interacted with ubiquinone- and phospholipid-depleted reductase, the spin label was totally immobilized. However, when phospholipids were replenished, the spin label showed mobility behaviour similar to that observed in a hydrophobic environment, indicating that the alkyl side chain of ubiquinone is extended into the hydrophobic region of intact reductase and has some degree of mobility.     

Association-dissociation of histone oligomers. A spin label study

The spin label method has been used to obtain information about conformational changes of histone oligomers taking advantage of the fact that at a low ionic strength and in the presence of other histones about 45% of cysteine residues of histone H3 react with the 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl spin label. For the labeled complexes H3-H4 and H nu the degree of immobilization of the spin label is a function of the ionic strength. This variation is identical for both complexes within a long range of ionic strengths, including the interval of 0.8-2 M NaCl, under which conditions interactions are known to exist between the tetramer (H3)2 (H4)2 and the dimer (H2A) (H2B). This finding suggests a negligible influence of the dimer for modifying the cysteine residue environment of histone H3 on octamer formation. GuHCl treatment at high ionic strength of the labeled complexes gives rise to a non-lineal increase in the degree of mobility of the spin label. This increase, at low GuHCl concentration (0-0.5 M GuHCl), is interpreted as showing a lowering in rigidity for the Cys residue environment, without affecting the general stability of the tetramer (H3)2 (H4)2. At higher GuHCl concentration (2-3 M GuHCl) the increase in the spin label mobility is related to a dissociation of the complexes in single histones. Our results are consistent with the view that the overall structure of the tetramer, as well as its conformational changes during complex structuration or denaturation, are not strongly affected by the presence of the dimer (H2A) (H2B).     

On the use of the spin labeling technique in the study of erythrocyte membranes.

ESR spectra and scanning electron micrographs of human erythrocytes spin labeled with the conventional stearic acid nitroxide substituted at the 5-position have been obtained over a range of label-to-lipid ratios. While morphological changes as previously reported (Bieri, V. G., Wallach, D. F. H. and Lin, P. S. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4797-4801) are reproduced, it is shown that at label-to-lipid ratios of 1:10 or less the basic ESR spectrum is not significantly affected. At low label concentrations the spin labeling technique is a viable one and can be used to investigate membrane properties.     

Tertiary structure variability within the quaternary states of hemoglobin: a spin label study

Using variable temperature techniques, the spin label spectral resolution of hemoglobin labeled at the beta93 cysteines with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodonacetamide has been greatly enhanced. The effects of different ligands, inositol hexaphosphate, pH and salt concentration upon spin labeled ferrous and ferric hemoglobin indicate that the beta chain tertiary structure exhibits considerable variability within the oxy and deoxy quaternary structures. From these studies ligand and spin state changes both appear to be of significance in producing structural changes; binding of inositol hexaphosphate then produces further structural changes secondary in amplitude.     

Electron spin resonance and fluorescence studies of the bound-state conformation of a model protein substrate to the chaperone SecB.

SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The electron spin resonance data suggest that these cysteine residues are in close proximity (15 A) when no substrate protein is bound but move away to a distance of greater than 20 A when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.     

Enzymatic sequence-specific spin labeling of a DNA fragment containing the recognition sequence of EcoRI endonuclease

Deoxyuridine analogs spin labeled in position 5 have been enzymatically incorporated sequence specifically into an oligodeoxyribonucleotide to form a spin-labeled 26-mer. The 26-mer contains the EcoRI-binding site and two labels which are located symmetrically close to the binding site. The labels are separated from one another far beyond the Heisenberg spin-exchange distance. The local base motion as determined by ESR spectroscopy is of the order of 4 ns in the oligonucleotide duplex. This is the same value as reported earlier for local T motions in polynucleotide duplexes, thereby providing direct experimental evidence that the ESR line shape of spin levels covalently attached to nucleic acids depends primarily on the local dynamics of the nucleic acid building blocks.     

Characterization of semiquinone free radicals formed from stilbene catechol estrogens. An ESR spin stabilization and spin trapping study

Electron spin resonance spectroscopy has been used to detect, characterize, and to infer structures of o-semiquinones derived from stilbene catechol estrogens. Radicals were generated enzymatically using tyrosinase and were detected as their Mg2+ complexes. It is suggested that initial hydroxylation of stilbene estrogen gives a catechol estrogen in situ; subsequent two-electron oxidation of the catechol to the quinone, followed by reverse disproportionation, leads to the formation of radicals. Consistent with this mechanism, o-phenylenediamine, a quinone trapping agent, inhibits formation of o-semiquinones. A competing mechanism of radical production involves autoxidation of the catechol. Hydroxyl radicals are shown to be produced in this system via a mechanism involving reduction of iron and copper complexes by stilbene catechols. Possible differences in the reactivity of stilbene ortho- and para-semiquinones are discussed.