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PubMed1990

Spin labeling of calcium-dependent phospholipid-binding proteins.

Bovine lung annexins p32 and p34 were spin labeled with an iodoacetamidoproxyl spin label, a reagent that reportedly couples with protein methionine residues. Labeling conditions and stoichiometry were studied with the radiolabeled analogue [1-14C]iodoacetamide. As judged by this method, carboxamidomethylation of both p32 and p34 occurred up to a 0.7 mol ratio after 60 h of reaction at 37 degrees C and at pH 4. The two proteins retained Ca2(+)-dependent phospholipid-binding ability both in radiolabeled and in spin-labeled forms. Electron resonance spectra of spin-labeled p32 and p34 showed the features of a partially immobilized spin probe, with rotational correlation time values of 1.15 and 1.25 ns, respectively, which definitely indicate successful spin labeling. Quantitation of ESR spectra by computer double integration indicated 70% spin labeling of both proteins, as anticipated by radiolabeling. The use of spin-labeled p32 and p34 in the study of Ca2(+)-dependent interaction of annexins with biomembranes is proposed.

Animals↗
PubMed1985

A spin label study of the ionic strength dependent conformational change in the human Ia molecule.

Purified human Ia molecules were labelled with maleimide or isothiocyanate spin labels or by reacting "TEMPAMINE" spin label with the neuraminic acid of their carbohydrate residues. It was found that increasing the ionic strength from 0.05 to 0.75 markedly increased the dipolar interaction between the maleimide-attached labels, but no effect was found of ionic strength or motion on dipolar interaction with the other two labels. The effect of increasing ionic strength could be blocked by the prior addition of Ia-specific antibody, but could not be reversed by the addition of antibody after ionic strength was increased. These findings complement an earlier finding that increasing ionic strength over the range 0.05-0.75 has an inhibitory effect on the combination of Ia with its antibody. Because the maleimide spin labels attach predominantly to SH groups it is suggested that increasing ionic strength causes conformational changes in the immunoglobulin loop region which alter the accessibility of the Ia antibody-binding site.

Antibody Specificity↗
PubMed1979

Synthesis of chromophoric, spin label enzyme substrates useful for cryoenzymology.

The spin label nitroxide derivative 3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oic acid has been synthesized and characterized by chemical methods. It is a useful intermediate in the preparation of a new class of chromophoric spin label substrates for enzyme studies, as shown by the synthesis of O-3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oyl-L-beta-phenyllactic acid, a specific ester substrate of bovine pancreatic carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2). Kinetic parameters of the esterolytic reaction are conveniently determined by UV spectrophotometric methods, and a reaction intermediate can be stabilized in fluid cryosolvent mixtures at subzero temperatures. Results are presented of preliminary electron spin resonance studies to demonstrate that structural relationships of the spin label substrate in a catalytically active configuration to active site residues can be determined for this low temperature-stabilized reaction intermediate. This substrate thus demonstrates the utility of this new class of spin label derivatives for characterization of enzyme reaction intermediates stabilized by cryoenzymologic techniques.

Animals↗
PubMed1992

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. This provides strong evidence that the helical conformation of these peptides is not stabilized by intermolecular interactions.

Alanine↗
PubMed1982

Fusion and lipid exchange in vesicles containing lipophilic spin labels.

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.

Liposomes↗
PubMed1973

Spin label motion in fatty acids.

Spin labels dissolved in highly purified fatty acid systems exhibit nearly identical tumbling rates in liquid and solid phases. Even though the spin labels do not have the same molecular geometry as the lipid matrix the melting point of the matrix can be inferred by measurements of the temperature dependency of molecular motion.

Chromatography, Thin Layer↗
PubMed1976

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.

Acetylcholine↗
PubMed2001

Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II.

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 A. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.

Carbonic Anhydrases↗
PubMed1993

Probes of membrane electrostatics: synthesis and voltage-dependent partitioning of negative hydrophobic ion spin labels in lipid vesicles.

Two spin-labeled derivatives of the hydrophobic anion trinitrophenol have been synthesized and characterized in lipid vesicles. In the presence of lipid vesicles, the electron paramagnetic resonance (EPR) spectra of these probes are a composite of both membrane-bound and aqueous populations; as a result, the membrane-aqueous partitioning can be determined from their electron paramagnetic resonance spectra. The effect of transmembrane potentials on the membrane-aqueous partitioning of these spin-labeled hydrophobic ions was examined in phosphatidylcholine vesicles formed by extrusion. Inside positive membrane potentials promote an increase in the binding of these probes that is quantitatively accounted for by a simple thermodynamic model used previously to describe the partitioning of paramagnetic phosphonium ions. The transmembrane migration rates of these ions are dependent on the dipole potential, indicating that these ions transit the membrane in a charged form. The partitioning of the probe is also sensitive to the membrane surface potential, and this dependence is accurately accounted for using the Gouy-Chapman Stern formalism. As a result of the membrane dipole potential, these probes exhibit a stronger binding and a more rapid transmembrane migration rate compared with positive hydrophobic ion spin labels and provide a new set of negatively charged hydrophobic ion probes to investigate membrane electrostatics.

Biophysical Phenomena↗
PubMed1992

Spin-labeling studies of rat liver NADPH-cytochrome p450 reductase: conformation and function relationship.

ESR spin-labeling studies designed to yield information regarding the relationship between function and conformation of rat liver NADPH-cytochrome P450 reductase (EC 1.6.4.2) were carried out. The purified enzyme was spin labeled by a nitroxide derivative of p-chloromercuribenzoate. Two conditions for spin labeling were employed: (i) the presence of NADP+, yielding an active site-protected spin-labeled reductase, and (ii) the absence of NADP+, yielding completely spin-labeled reductase. Reductase in which the active site was protected by binding NADP+ and then spin-labeled retains most of its enzymatic activity; on the other hand, completely spin-labeled reductase is devoid of any enzymatic activity. Completely spin-labeled reductase yields a two-component resolved ESR spectrum that reflects two classes of spin-labeled binding sites, a strongly immobilized (S) and a weakly immobilized (W) site. The ratio of W/S provides a valuable parameter for studying the relationship between function and conformation. Structural perturbants, such as urea, KCl, and pH, were employed to determine their effects on the activity of the enzyme and their relationship to changes in the conformational state of the reductase. It was further observed that the enzymatically active spin-labeled derivative generated superoxide radical in the presence of NADPH and cytochrome c, which in turn reduced completely the attached spin-label.

Acetylation↗
PubMed1975

Interaction between spin-labeled acyl-coenzyme A and the mitochondrial adenosine diphosphate carrier.

Spin-labeled long-chain (m,n)acyl-CoA's (general formula: CH3(CH2)mCR(CH2)nCOSCoA, where R is an oxazolidine ring containing a nitroxide) inhibit anion transports through the inner mitochondrial membrane at low concentrations as ordinary long-chain acyl-CoA's do. The inhibition constant relative to the inhibition of the ADP transport in heart mitochondria by spin-labeled palmityl-CoA and stearyl-CoA is of the order of 10-7 M, a value which is similar to that found for natural long-chain acyl-CoA's. A short-chain spin-labeled acyl-CoA (C5) showed no inhibitory effect in the range of concentrations tested (up to 30 muM). (2) (10,3)Acyl-CoA added to heart mitochondria at low concentrations exhibits spectra corresponding to an immobilized probe. The corresponding free fatty acid shows a higher freedom of motion between 0 and 30 degrees. The same differences in spectra of spin-labeled acyl-CoA and spin-labeled free fatty acid were found in inner membrane vesicles from rat liver mitochondria, but not in outer membrane preparations. (3) The selective interaction of spin-labeled acyl-CoA with the ADP carrier is indicated by the release of this interaction by specific ligands of the ADP carrier, such as ADP or ATP, carboxyatractyloside, adn bongkrekic acid. ADP (or ATP) and carboxyatractyloside rendered the spin-labeled (10,3)acyl-CoA nearly as mobile as the (10,3) free fatty acid. No effect was obtained with AMP, GDP, or UDP which are not transported by the ADP carrier. Bongkrekic acid, another specific inhibitor of the ADP carrier, was inactive when added alone; however, it was effective when added together with amounts of ADP which are ineffective per se. (4) The electron spin resonance (esr) spectrum observed at low concentrations of (10,3)acyl-CoA arises from (10,3)acyl-CoA bound to the ADP carrier. At higher concentrations the (10,3)-acy-CoA is more suggesting that the bulk of the label is also present in the lipid phase of the membrane. Spin-labeled acylCoA's incorporated into a sonicated dispersion of lipids extracted from heart mitochondria exhibited similar mobile spectra. (5) When the oxazolidine ring is moved down the hydrocarbon chain of the acyl-CoA, the binding features tended to disappear. Whereas nitroxide-protein interactions could be easily measured with the (10,3)acyl-CoA and the (7,6)acyl-CoA, much less or even no significant interactions could be detected with the (5,10)acyl-CoA or the (1,14)acyl-CoA. (6) The above results suggest that spin-labeled long-chain acylCoA added to mitochondria binds by its polar moiety to the ADP carrier. The acyl chain interacts with the ADP carrier protein over a length of 10-15 A. The remaining portion of the acyl chain experiences a fluid lipid environment.

Adenosine Diphosphate↗
PubMed1976

Motion of spin-labeled fatty acids in murine macrophages. Relation to cellular phagocytic activity.

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels. Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings (2 T11) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives. Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 degrees C, or by fixing them with 0.25% glutaraldehyde. No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with cis- or trans-unsaturated fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.

Animals↗
PubMed1983

Collisions between nitrogen-14 and nitrogen-15 spin-labels. 2. Investigations on the specificity of the lipid environment of rhodopsin.

The method of spin-spin interactions between 15N and 14N spin-labels was used to investigate lipid-protein collision rates in reconstituted vesicles containing rhodopsin from bovine disk membranes and an equimolar mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In each sample, a fraction of one of the three phospholipids was labeled with 14N spin-label while a 15N spin-labeled fatty acid was covalently linked to rhodopsin. The extent of spin-spin interaction between 15N and 14N labels was either calculated by complete spectral simulation or evaluated from the line broadening as deducted from the intensity decrease of the low-field 15N line. It was found that all three spin-labeled phospholipids utilized for these experiments can interact magnetically with the spin-labeled rhodopsin. Above 35 degrees C little difference between the three species can be detected. Calculation of the diffusion constant of the phospholipids at the boundary of rhodopsin proves that the lifetime of the phospholipids at the protein boundary is short and that no long-lived annular lipids are segregated. At temperatures below approximately 30 degrees C the spectra of the samples containing spin-labeled phosphatidylserine depend upon the presence or absence of calcium. The extent of 15N line broadening was found weaker in the presence of Ca2+ than in the presence of ethylenediaminetetraacetate. Thus Ca2+ tends to exclude phosphatidylserine from the lipid environment of rhodopsin. This observation can be attributed to the formation of specific lipid domains within the membrane, induced by Ca2+.

Animals↗
PubMed1976

Synthesis and biological properties of some spin-labeled 9-aminoacridines.

Five spin-labeled 9-aminoacridines, each bearing either a 4-(2,2,6,6-tetramethyl-1-piperidinyloxy) or a 3-(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety in the 9 position, have been synthesized and assayed for biological activity in three different test systems. Sedimentation velocity measurements indicated that the labels caused unwinding of calf thymus DNA. Those acridines which contained both 6-chloro and 2-methoxy substituents were less toxic to leukemia L1210 in static culture than the corresponding unsubstituted analogues. While the unsubstituted aminoacridines were quite good inhibitors of Escherichia coli DNA-primed RNA polymerase, the 6-chloro-2-methoxy-substituted compounds stimulated this enzyme system. In the presence of E.coliDNA, the ESR spectrum of 4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2,2,6,6-tetramethyl-1-piperidinyloxyl (12) became broad and highly asymmteric with a maximal hyperfine splitting of 57.5 G. This observation suggests that when 12 intercalates into DNA the piperidinyl moiety that bears the nitroxide group becomes highly immobilized. These results suggest that the spin-labeled 9-aminoacridines will be useful probes for nucleic acids.

Acridines↗
PubMed1990

Assignment of proton endor resonances of nitroxyl spin-labels in frozen solution.

Spin-label nitroxyl derivatives of tetramethylpyrroline and tetramethylpyrrolidine in frozen solutions of perdeuterated methanol have been characterized by electron nucleus double resonance (ENDOR spectroscopy). With use of selectively deuterated derivatives of 2,2,5,5-tetramethylpyrroline-1-oxyl-3-carboxamide, proton ENDOR resonance features have been assigned to the vinylic proton in the five membered pyrrolinyl ring and to the methyl groups. The ENDOR resonance features were analyzed on the basis of their dependence on H0. Two pairs of resonance features were assigned to the vinylic proton and were shown to correspond to parallel and perpendicular hyperfine coupling (hfc) components. Six pairs of resonance features were ascribed to the methyl groups. The proton ENDOR spectra of the 3-carboxylic acid spin-label derivatives of tetramethylpyrroline and of tetramethylpyrrolidine compounds exhibited comparable features with nearly identical line splittings. From the observed ENDOR splittings, we have estimated the isotropic hfc component of the vinylic proton in 2,2,5,5-tetramethylpyrroline-1-oxyl-3-carboxamide to be -1.81 +/- 0.04 MHz in frozen methanol. On the basis of the anisotropic dipolar hfc components, the electron-to-vinylic proton distance is estimated as 3.78 +/- 0.01 A, in excellent agreement with that of 3.79 A calculated from X-ray defined coordinates.

Cyclic N-Oxides↗
PubMed1993

Protein rotational dynamics investigated with a dual EPR/optical molecular probe. Spin-labeled eosin.

An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in studies of rotational dynamics of the free probe and of the probe when bound noncovalently to bovine serum albumin using the spectroscopic techniques of fluorescence anisotropy decay and electron paramagnetic resonance (EPR) and their long-lifetime counterparts phosphorescence anisotropy decay and saturation transfer EPR. Previous work (Beth, A. H., Cobb, C. E., and J. M. Beechem, 1992. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe. Society of Photo-Optical Instrumentation Engineers. Time-Resolved Laser Spectroscopy III. 504-512) has shown that the spin-label moiety only slightly altered the fluorescence and phosphorescence lifetimes and quantum yields of 5-SLE when compared with 5-SLE whose nitroxide had been reduced with ascorbate and with the diamagnetic homolog 5-acetyleosin. In the present work, we have utilized time-resolved fluorescence anisotropy decay and linear EPR spectroscopies to observe and quantitate the psec motions of 5-SLE in solution and the nsec motions of the 5-SLE-bovine serum albumin complex. Time-resolved phosphorescence anisotropy decay and saturation transfer EPR studies have been carried out to observe and quantitate the microseconds motions of the 5-SLE-albumin complex in glycerol/buffer solutions of varying viscosity. These latter studies have enabled a rigorous comparison of rotational correlation times obtained from these complementary techniques to be made with a single probe. The studies described demonstrate that it is possible to employ a single molecular probe to carry out the full range of fluorescence, phosphorescence, EPR, and saturation transfer EPR studies. It is anticipated that "dual" molecular probes of this general type will significantly enhance capabilities for extracting dynamics and structural information from macromolecules and their functional assemblies.

Biophysical Phenomena↗
PubMed1979

Ternary complex formation of pig heart lactate dehydrogenase with spin-labelled coenzyme and inhibitors as studied by electron spin resonance.

The formation of ternary inhibitor and 'dead end' complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) was studied by means of two NAD derivatives, spin-labelled at N6 and C-8 of the adenine ring. Dissociation constants calculated for the inhibitors oxamate and oxalate from their corresponding ternary complexes are in excellent agreement with data from literature derived from sedimentation experiments. However, the recently postulated enzyme-NADH-sulfite complex was not observed. The mobility of the spin-label, i.e. the protein conformation near the adenine binding pocket in various ternary complexes depends on the type of inhibition or substrate employed.

Animals↗
PubMed2000

Dynamics and ordering in a spin-labeled oligonucleotide observed by 220 GHz electron paramagnetic resonance.

The dynamics of a newly synthesized cytosine spin-label and the spin-labeled pentamer TTC*TT have been observed by high-frequency (220 GHz) electron paramagnetic resonance (EPR) in aqueous solution at ambient temperature using only nanomolar amounts of spin-label. Temperature studies were carried out for both labeled species in buffer containing glycerol. The motion of the spin-labeled monomer could be fitted using a model of fully anisotropic rotation (FAR) over the entire temperature range studied. In the single-stranded pentamer, the high-field spectra are best interpreted using a model of microscopic ordering with macroscopic disorder (MOMD) with the probe in a highly nonpolar environment. The observed local order parameters of 0.60-0.70 suggest a micelle-like structure in which the label is tightly packed with the hydrophobic bases. These preliminary studies illustrate how the excellent orientation selectivity of high-field EPR provides new dynamic information about local base motions in DNA, and also how high-field EPR of spin-labels allows one to discriminate accurately between the effects of local versus global motions in spin-labeled macromolecules.

Base Sequence↗