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 1,063 records · Page 59Linked to original sources
PubMed1989

Spin-labeled analogues of ATP, ADP and AMP: substitutes for normal nucleotides in biochemical systems.

The different roles and effectiveness of adenosine monophosphate, diphosphate and triphosphate labeled at the 6 position of the purine ring with 2,2,6,6-tetramethylpiperidine-1-oxyl in reactions catalyzed by Escherichia coli glutamine synthetase (GS) have been investigated. Our results show that the spin-labeled ATP (Tempo-ATP) serves as a substrate in the glutamine synthesis reaction and in the adenylation of E. coli glutamine synthetase catalyzed by ATP: glutamine adenylyl transferase (ATase) with essentially the same effectiveness as normal ATP. In another reaction (gamma-glutamyltransferase), Tempo ADP serves as an effector with a Km of 9.4 . 10(-8) M compared to 1.2 . 10(-8) M for the normal ADP, while covalently bonded Tempo-AMP serves as a modifier on the catalytic properties of E. coli glutamine synthetase just as the covalently bonded normal AMP does. The dissociation constants between the labeled nucleotides, Mn2+, Mg2+ and Ca2+ are in the same order of magnitude as the binding constants for those cations and the corresponding normal nucleotides. Our findings indicate that the spin-labeled nucleotides are good substitutes for the normal nucleotides in the biochemical systems studied.

Adenosine Diphosphate↗
PubMed1999

EPR mapping of interactions between spin-labeled variants of human carbonic anhydrase II and GroEL: evidence for increased flexibility of the hydrophobic core by the interaction.

Human carbonic anhydrase II (HCA II) interacts weakly with GroEL at room temperature. To further investigate this interaction we used electron paramagnetic resonance (EPR) spectroscopy to study HCA II cysteine mutants spin-labeled at selected positions. From our results it is evident that protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements. HCA II needs to be unfolded to about the same extent as a GuHCl-induced molten-globule intermediate of the enzyme to interact with GroEL. The interaction with GroEL includes interactions with outer parts of the HCA II molecule, such as peripheral beta-strands and the N-terminal domain, which have previously been shown to be rather unstable. As a result of the interaction, the rigid and compact hydrophobic core exhibits higher flexibility than in the molten globule, which is likely to facilitate rearrangements of misfolded structure during the folding process. The degree of binding to GroEL and accompanying inactivation of the enzyme depend on the stability of the HCA II variant, and nonspecific hydrophobic interactions appear to be most important in stabilizing the GroEL-substrate complex.

Amino Acid Substitution↗
PubMed1983

A spin label study of the urinary bladder luminal membrane.

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.

Animals↗
PubMed1994

Selective cysteine-->serine replacements in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens allow the unambiguous assignment of Cys211 as the site of modification by spin-labeled p-chloromercuribenzoate.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains five sulfhydryl groups per subunit. Cysteine-->serine replacements show that the thiols are not essential for catalysis. The increased dissociation constant for FAD in mutant Cys158Ser suggests that Cys158 is important for the solvation of the pyrophosphate moiety of the prosthetic group. Wild-type p-hydroxybenzoate hydroxylase is rapidly inactivated by mercurial compounds. Inactivation by a spin-labeled derivative of p-chloromercuribenzoate is fully abolished in mutant Cys211Ser. Incorporation of the spin label in the other Cys-->Ser mutants strongly impairs substrate binding without affecting the catalytic properties of the FAD. The results are discussed with respect to previous tentative assignments from chemical modification studies and in light of the 3-D structure of the enzyme-substrate complex.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1987

Studies of the active site of cytochrome P-450scc with a high-affinity spin-labeled inhibitor.

The intramolecular site of P-450scc for conversion of cholesterol to pregnenolone involves a substrate site, an active site, and a site for transmission of electrons. The substrate site was studied with a high-affinity, high-potency nitroxide spin-labeled inhibitor of cholesterol side-chain cleavage. This substance, 17 alpha-hydroxy-11-deoxycorticosterone nitroxide (SL-V), has an affinity comparable to that of the most active substrate inhibitors ever reported and 2-50 times greater than that of the natural substrate cholesterol. Competition experiments with cholesterol and its analogues confirmed that SL-V binds reversibly to the substrate site. Titration experiments showed a single binding site on the P-450 molecule. The substrate site is on the apoprotein and has little or no direct interaction with the heme. Spin-spin interactions between the Fe3+ and side-chain or A-ring spin-labeled groups could not be demonstrated, which is consistent with carbons 22 and 20 being closest to the heme iron. We postulate that substrate disrupts a histidine nitrogen coordination with the heme iron and induces conformational changes in the apoprotein. These changes lead to increased affinity for iron-sulfur protein.

Affinity Labels↗
PubMed1982

Immunogenicity of liposomal model membranes sensitized with spin-labeled haptens and topographical distribution of haptens on the membranes.

The relation between the in vitro immunogenicity of phosphatidylcholine liposomes containing 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) as a hapten and the topographical distribution of the haptens on lipid membranes was studied. In distearoylphosphatidylcholine liposomes, the immunogenicity increased with increase of cholesterol content in the liposomal membranes. The electron spin resonance spectra of spin-labeled DNP-Cap-PE in distearoylphosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled DNP-Cap-PE on the membranes. In the absence of cholesterol, a considerable amount of haptens was clustered on the distearoylphosphatidylcholine membranes, but with increase of cholesterol, random distribution of the haptens on the membranes increased. The cholesterol-dependent change in the topographical distribution of the haptens on the membranes paralleled the change of immunogenicity, i.e., the immunogenicity was low when haptens were clustered on the liposomal membranes. Haptens arranged at a proper distance on the membranes may be required for optimum immune response.

Animals↗
PubMed1999

Interactions between lipid-anchored and transmembrane proteins. Spin-label ESR studies on avidin-biotinyl phosphatidylethanolamine in membrane recombinants with myelin proteolipid proteins.

Interactions between lipid-anchored and transmembrane proteins are relevant to the intracellular membrane sorting of glycosyl phosphatidylinositol-linked proteins. We have studied the interaction of a spin-labeled biotinyl diacyl phospholipid, with and without specifically bound avidin, with the myelin proteolipid protein (or the DM-20 isoform) reconstituted in dimyristoylphosphatidylcholine. Tetrameric avidin bound to the N-biotinyl lipid headgroup is a surface-anchored protein, and the myelin proteolipid is an integral protein containing four transmembrane helices. The electron spin resonance (ESR) spectrum of N-biotinyl phosphatidylethanolamine spin-labeled at the C-14 position of the sn-2 chain consists of two components in fluid-phase membranes of dimyristoylphosphatidylcholine containing the proteolipid. In the absence of avidin, this is characteristic of lipid-protein interactions with integral transmembrane proteins. The more motionally restricted component represents the lipid population in direct contact with the intramembranous surface of the integral protein, and the more mobile component corresponds to the bulk fluid lipid environment of the bilayer. In the presence of avidin, the biotin-lipid chains have reduced mobility because of the binding to avidin, even in the absence of the proteolipid [Swamy, M. J., and Marsh, D. (1997) Biochemistry 36, 7403-7407]. In the presence of the proteolipid, the major fraction of the avidin-anchored chains is further restricted in its mobility by interaction with the transmembrane protein. At a biotin-lipid concentration of 1 mol %, approximately 80% of the avidin-linked chains are restricted in membranes with a phosphatidylcholine:proteolipid molar ratio of 37:1. This relatively high stoichiometry of interaction can be explained when allowance is made for the closest interaction distance between the lipid-anchored avidin tetramer and the transmembrane proteolipid hexamer, without any specific interaction between the two types of membrane-associated proteins. The interaction is essentially one of steric exclusion, but the lipid chains are rendered more sensitive to interaction with the integral protein by being linked to avidin, even though they are removed from the immediate intramembrane protein-lipid interface. This could have implications for the tendency of lipid-anchored chains to associate with membrane domains with reduced lipid mobility.

Animals↗
PubMed2006

TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes.

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.

Alamethicin↗
PubMed2005

Peptide-lipid interaction monitored by spin labeled biologically active melanocortin peptides.

The present work comparatively analyzes the interaction of alpha-MSH and its more potent and long-acting analog [Nle4, D-Phe7]alpha-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac0 and the time resolved fluorescence of Trp9 present in the peptides. The Toac0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pKa 7.5, possibly that of His6, can be clearly monitored by peptide-lipid partition. Trp9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone.

Electron Spin Resonance Spectroscopy↗
PubMed2004

Polydisperse composition of mixed monolayer-protected, spin-labeled Au nanoparticles.

Using EPR spectroscopy and spin-labeled ligands, we have studied product distribution in the ligand exchange reactions of Au nanoparticles. When the incoming ligand was used in excess, the exchanged nanoparticles had uniform composition of the organic shell. Preparative GPC was employed to fractionate these materials according to the diameter of the Au core, as confirmed by TEM, UV, and EPR measurements. When the exchange reaction was carried out with less than a stoichiometric amount of the incoming ligand, nanoparticles with polydisperse composition of the organic shell were formed. This was established by the EPR analysis of the exchange reaction mixtures following their GPC fractionation. The GPC separation of these mixtures is controlled by both Au core size and shell composition.

Journal Article↗
PubMed1979

Effects of spin-labeled stearates on duck erythrocyte adenylate cyclase.

The effects of various spin-labeled stearates on duck erythrocyte adenylate cyclase were investigated. Only 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl caused an increase in adenylate cyclase activity. It increased the basal rate by about 50%, and the activities stimulated by isoproterenol and isoproterenol plus guanosine 5'-[beta,gamma-imido]triphosphate by 35%. Upon analysis of the width parameter delta1 in the electron spin resonance spectra for both the basal enzyme activity and the stimulation obtained with effectors such as guanosine 5'-[beta,gamma-imido]triphosphate, isoproterenol, isoproterenol plus guanosine 5'-[beta,gamma-imido]triphosphate and NaF, a correlation of the changes of modification in adenylate cyclase activities was found. These findings suggest that the molecular environment of the enzyme has been modified.

Adenylyl Cyclases↗
PubMed1994

Binding and state of aggregation of spin-labeled cecropin AD in phospholipid bilayers: effects of surface charge and fatty acyl chain length.

The binding and state of aggregation of cecropin in large unilamellar vesicles of different surface potential and varying acyl chain length were examined using a Cys-33 spin-labeled derivative of cecropin AD (CAD). Association isotherms of the peptide were measured for vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) containing 5, 15, and 30 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). The isotherms display a concentration-dependent positive cooperativity indicating the possible formation of cecropin aggregates in the lipid phase. The critical aqueous concentration for aggregation was dependent on the fraction of POPG, suggesting the involvement of acidic lipids in the formation and stabilization of the putative aggregate. Our data also indicate that cooperativity depends on the state of side-chain ionization of an acidic residue that titrates between pH 7 and 4.4. The binding of spin-labeled Cys-33 CAD was found to be influenced by the acyl chain length of the host lipid. The association isotherm of the peptide for dilaureoyl-sn-glycero-3-phosphatidylcholine vesicles containing 30 mol % dilauroyl-sn-glycero-3-phosphatidylglycerol (DLPG) differed significantly from that in POPC/POPG and could be interpreted in terms of a monomer-monomer partitioning between the aqueous and lipid phases. ESR line-shape analysis was consistent with peptide aggregation in dioleoyl-sn-glycero-3-phosphatidylglycerol vesicles but not in DLPG vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1978

Spin label detection of intermolecular interactions in carbonmonoxy sickle hemoglobin.

With recently developed spin label techniques for monitoring macromolecular rotational motion, heme-liganded sickle cell hemoglobin in the presence of inositol hexaphosphate is shown to exhibit restricted motional freedom as compared to liganded normal adult human hemoglobin. This motional restriction is dependent on both hemoglobin concentration and temperature.

Adult↗
PubMed1980

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.

Actins↗
PubMed1982

Convenient biosynthetic preparation of isomeric spin-labelled radioactive phosphatidic acids.

A convenient method for the enzymatic preparation of sn-3-[2-3H]phosphatidic acids carrying also 5-, 12-, or 16-nitroxide stearic acids, from sn-3-[2-3H]glycerophosphate and isolated guinea pig liver microsomes, is described in detail. The procedure allows a simultaneous preparation of three spin-labelled sn-3-[2-3H]phosphatidic acids of yields 3-3.5 mumol of each compound which is greater than 99% pure in respect to the radioactivity and which contains 25 mol% of spin-labelled fatty acids. These phosphatidic acids were approximately equally distributed between the primary and the secondary hydroxyl when 12- or 16-nitroxide stearic acids were used or predominantly (75%) associated with the secondary hydroxyl of sn-3-[2-3H]phosphatidic acid when 5-nitroxide stearic acid was present in the incubation mixture.

Animals↗
PubMed1987

A long chain spin label for glycosphingolipid studies: transbilayer fatty acid interdigitation of lactosyl ceramide.

16-Carbon and 18-carbon fatty acids with covalently attached nitroxide free radicals have seen wide usage in membrane studies of phospholipid dynamics, orientation, and associations. However, they are inadequate for dealing with some very important questions that relate to glycosphingolipids. We report here the synthesis of a long chain (24-carbon) spin-labelled fatty acid designed for such problems. We have used both the new 24-carbon and the more conventional 18-carbon spin-labelled fatty acids to replace the natural fatty acid of lactosyl ceramide so that we may begin to compare short and long chain derivatives to analyse the molecular basis of their functional differences. Spectra seen are consistent with the view that in a bilayer host matrix the methyl end of the long fatty acid crosses the hydrophobic membrane center and interdigitates with fatty acids of phospholipids of the opposing monolayer.

Antigens, CD↗
PubMed1977

Studies on spin-labelled egg lecithin dispersions.

ESR spectra of egg lecithin dispersions labelled with 5-nitroxide stearic acid are recorded with a 50 G field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The expansion technique improves the precision of the order parameters determined from the hyperfine splitting measurements, and may prove useful in future spin label membrane studies. Approximate order parameters are derived to describe the fluidity of fatty acid spin-labelled membranes in those cases where either the inner or outer hyperfine extrema are not well defined. The ability of these expressions to measure the fluidity of labelled egg lecithin disperions for the temperature range 14-42 degrees C is examined.

Egg Yolk↗