A study has been made of the interaction of specific anti-nitroxide (anti-spin label) IgG antibodies, Fab fragments, complement, and liposomes containing dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and dipalmitoylphosphatidylcholine plus 0-50 mole % cholesterol, together with various concentrations of a head group spin-labeled phospholipid (0.5-0.01 mole % in the plane of th...

A modified and improved spin membrane immunoassay has been developed for detecting complement-activating antibodies to Neisseria meningitidis capsular polysaccharide antigens. The polysaccharides were incorporated in the membranes of large unilamellar vesicles prepared by the reverse-phase evaporation method and filled with the water-soluble spin label tempocholine chloride. Upon addition of gr...

The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near-full-length EGFR, we determined defined distance distributi...

Spin labels have now enjoyed a history of close to twenty-five years in demonstrating the applicability of paramagnetic nitroxides to biochemical problems of structure and function in enzymes, membranes, cells and animals. These have been employed traditionally as reporter groups; that is, the nitroxide spin label serves as a physical probe which reports aspects of its structure and environment...

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has lo...

This report describes a method suitable for determining the depth of a wide variety of fluorescent molecules embedded in membranes. The method involves determination of the parallax in the apparent location of fluorophores detected when quenching by phospholipids spin-labeled at two different depths is compared. By use of straightforward algebraic expressions, the method allows calculation of d...

Maleimide spin label was covalently bound to sulfhydryl residues of D-amino acid oxidase and lipoamide dehydrogenase. Labeling of native D-amino acid oxidase resulted in a non-homogeneous EPR-spectrum, which consisted of 4 moles of spin label bound immobile to the enzyme (mol.-weight 90.000). A detailed analysis of the spectrum, the kinetics of the reaction of the spin label with the protein an...

To test for possible differences in local conformation and S1 flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS) and a spin label having iodoacetamide reactivity. The marked activation of the Ca2+-ATPase (6- to 8-fold) and inhibition of the K+ (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH1) of...

We have investigated self-assembled stearic acid films adsorbed on an Al2O3 film by means of electron paramagnetic resonance (EPR) as well as near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. The stearic acid molecules are orientated in the film as shown by the NEXAFS data. Doping of the film with spin-labels in order to make the films accessible to EPR spectroscopy distorts the ...

We report a general approach for the simulation of the electron paramagnetic resonance (EPR) spectra of spin labels attached to peptides and proteins directly from replica-exchange molecular dynamics (REMD) trajectories. Conventional MD trajectories are generally inadequate for the prediction of EPR line shapes since the label can become trapped in one or more of a set of rotameric states, thus...