Soluble Spin Labels Research Articles

Structural Analysis of Large Protein Complexes Using Solvent Paramagnetic Relaxation Enhancements

The solvent helps out: Conventional protocols for structural analysis of protein complexes often fail if only sparse experimental data are available. NMR-spectroscopy-derived solvent paramagnetic relaxation enhancements (PREs) from a soluble spin label (picture) significantly improve the structural quality of a 150 kDa protein complex. This general protocol is an efficient approach for structural analysis of large protein complexes in solution.

On the surface properties of oleate micelles and oleic acid/oleate vesicles studied by spin labeling

Dilute aqueous systems composed of sodium oleate micelles and sodium oleate/oleic acid vesicles were investigated as a function of pH by electron spin resonance spectroscopy with TEMPO-stearate TEMPO-stearamide as well as with a positively charged water soluble spin label, TEMPO-choline. The dynamics of the three TEMPO-spin labels were found to be sensitive to changes in the interfacial region of the aggregates as a function of pH. The results obtained are consistent with the formation of a hydrogen bond network (RCOO−↔HOOCR) at the surface of the sodium oleate/oleic acid system in the course of the transformation of micelles into the closed bilayers (vesicles). Vesicles formation below pH 10 was determined independently with a spin labeled glucose derivative.

NMR Studies of Protein Hydration and TEMPOL Accessibility

Understanding the mechanisms of the interaction between a protein surface and its outer molecular environment is of primary relevance for the rational design of new drugs and engineered proteins. Protein surface accessibility is emerging as a new dimension of Structural Biology, since NMR methods have been developed to follow how molecules, even those different from physiological ligands, preferentially approach specific regions of the protein surface. Hen egg-white lysozyme, a paradigmatic example of the state of the art of protein structure and dynamics, has been selected as a model system to study protein surface accessibility. Bound water and soluble spin-labels have been used to investigate the interaction of this enzyme, both free and bound to the inhibitor (NAG) 3, with its molecular environment. No tightly bound water molecules were found inside the enzyme active site, which, conversely, appeared as the most exposed to visits from the soluble paramagnetic probe TEMPOL. From the presented set of data, an integrated view of lysozyme surface accessibility towards water and TEMPOL molecules is obtained.

NMR studies of protein surface accessibility.

Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity.

Spin-labeling study of membranes in wheat embryo axes. 1. Partitioning of doxyl stearates into the lipid domains

The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels ( n-SASL) and their methylated derivatives ( n-MeSASL), labelled at different positions of their doxyl group ( n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from −50 to +20°C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels.

An EPR investigation of surfactant action on bacterial membranes.

The effects of the surfactants, alcohol ethoxylate, amine ethoxylate, amine oxide and SDS on cell membranes were investigated using the lipid soluble spin label 5-doxyl stearic acid (5-DS). Electron paramagnetic resonance (EPR) spectroscopy revealed that the action of the surfactants was to significantly increase membrane fluidity of Proteus mirabilis, Staphylococcus aureus and Saccharomyces cerevisiae. The action of these surfactants as biocides was investigated and found to be dependent on the type of organism tested. There was, however, no direct correlation between enhanced membrane fluidity observed due to the action of the surfactants and biocidal activity. Data presented suggest that perturbing the fluidity of the cytoplasmic membrane is not immediately responsible for cell death.

An EPR investigation of surfactant action on bacterial membranes

The effects of the surfactants, alcohol ethoxylate, amine ethoxylate, amine oxide and SDS on cell membranes were investigated using the lipid soluble spin label 5-doxyl stearic acid (5-DS). Electron paramagnetic resonance (EPR) spectroscopy revealed that the action of the surfactants was to significantly increase membrane fluidity of Proteus mirabilis, Staphylococcus aureus and Saccharomyces cerevisiae. The action of these surfactants as biocides was investigated and found to be dependent on the type of organism tested. There was, however, no direct correlation between enhanced membrane fluidity observed due to the action of the surfactants and biocidal activity. Data presented suggest that perturbing the fluidity of the cytoplasmic membrane is not immediately responsible for cell death.

Tendamistat surface accessibility to the TEMPOL paramagnetic probe.

TEMPOL, the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, has been used to determine the surface characteristics of tendamistat, a small protein with a well-characterised structure both in solution and in the crystal. A good correlation has been found between predicted regions of exposed protein surface and the intensity attenuations induced by the probe on 2D NMR TOCSY cross peaks of tendamistat in the paramagnetic water solution. All the high paramagnetic effects have been interpreted in terms of more efficient competition of TEMPOL with water molecules at some surface positions. The active site of tendamistat coincides with the largest surface patch accessible to the probe. A strong hydration of protein N and C termini can also be suggested by this structural approach, as these locations exhibit reduced paramagnetic perturbations. Provided that the solution structure is known, the use of this paramagnetic probe seems to be well suited to delineate the dynamic behaviour of the protein surface and, more generally, to gain relevant information about the molecular presentation processes.

An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C

The paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO), was used to probe the surface exposure of methionine residues of recombinant cardiac troponin C (cTnC) in the absence and presence of Ca2+ at the regulatory site (site II), as well as in the presence of the troponin I inhibitory peptide (cTnIp). Methyl resonances of the 10 Met residues of cTnC were chosen as spectral probes because they are thought to play a role in both formation of the N-terminal hydrophobic pocket and in the binding of cTnIp. Proton longitudinal relaxation rates (R1's) of the [13C-methyl] groups in [13C-methyl]Met-labeled cTnC(C35S) were determined using a T1 two-dimensional heteronuclear single- and multiple-quantum coherence pulse sequence. Solvent-exposed Met residues exhibit increased relaxation rates from the paramagnetic effect of HyTEMPO. Relaxation rates in 2Ca(2+)-loaded and Ca(2+)-saturated cTnC, both in the presence and absence of HyTEMPO, permitted the topological mapping of the conformational changes induced by the binding of Ca2+ to site II, the site responsible for triggering muscle contraction. Calcium binding at site II resulted in an increased exposure of Met residues 45 and 81 to the soluble spin label HyTEMPO. This result is consistent with an opening of the hydrophobic pocket in the N-terminal domain of cTnC upon binding Ca2+ at site II. The binding of the inhibitory peptide cTnIp, corresponding to Asn 129 through Ile 149 of cTnI, to both 2Ca(2+)-loaded and Ca(2+)-saturated cTnC was shown to protect Met residues 120 and 157 from HyTEMPO as determined by a decrease in their measured R1 values. These results suggest that in both the 2Ca(2+)-loaded and Ca(2+)-saturated forms of cTnC, cTnIp binds primarily to the C-terminal domain of cTnC.

Soluble Spin-Label as a NMR Relaxation Probe in the Study of the Enzyme-Substrate Complexes

Abstract The paramagnetic contributions induced by TEMPOL soluble spin-label on gentiobiose carbon spin-lattice relaxation rates are analyzed. Selective effects in the β(1–6) glycosidic bond region were observed. The possibility of using soluble spin-label to determine the stereochemistry of the substrate-enzyme interaction was then explored. The results obtained with different diamagnetic and paramagnetic systems enabled us to distinguish the region of gentiobiose most involved in interaction with the enzyme, and the region of the disaccharide molecule located on the surface of the enzyme and most exposed to the nitroxide. The results obtained could be used to model the enzyme surface of the gentiobiose binding site. Soluble spin-labels have been widely used in the investigation of the conformational and dynamical properties of biomolecules in solution (1–4). The main effects of nit oxide spin-labels, like TEMPO or TEMPOL, is the increase in NMR spin-lattice relaxation rates of solvent exposed nuclei. In...

A 1H NMR study on the interaction of aminoxyl paramagnetic probes with unfolded peptides

The use of soluble spin labels to filter out the cross peaks of outer proton nuclei in 2D NMR spectra has been proposed as a general method to obtain structural information for complex molecules. Here the paramagnetic effects observed on backbone protons of an unfolded 27 amino acid peptide are discussed. The lack of any differential intensity change of the NH–Hα cross-peaks in TOCSY spectra is suggested as an additional general criterion for the identification of unfolded structures.

Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra: Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering

Soluble spin labels, which “bleach” the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed.

NMR delineation of inner and outer protons from paramagnetic relaxation perturbations in 1 D and 2D spectra of peptides

The use of soluble spin labels to filter out the cross-peaks of outer proton nuclei in 2D NMR spectra is proposed as a general method of obtaining structural information about complex molecules. Gramicidin S, a decapeptide of well defined and stable structure, has been used as a model system to correlate the paramagnetic effects observed in 1 D and 2D spectra and the peptide solution conformation. The method appears particularly suited to obtaining information about the hydrogen bonding of backbone amide protons.

Cytotoxicity of commonly used nitroxide radical spin probes

Since nitroxide radical spin probes are used frequently to test biophysical properties of cells, their use should be restricted to conditions that do not perturb normal cell growth and viability. Eight commonly used nitroxide radical spin probes have been tested for their effects on the survival of CHO cells. These include water-soluble spin probes Tempol, Tempamine, CTPO, CTPC and 4-maleimido-Tempo, and lipid soluble spin probes 5-Doxyl-, 12-Doxyl-, and 16-Doxylstearates. With the exception of 4-maleimido-Tempo, none of the water soluble spin labels inhibited cell survival at concentrations as high as 1 mM. At concentrations of 75 μM and higher, 4-maleimido-Tempo inhibited cell survival in a dose dependent manner. At concentrations commonly used for spin labeling of cells (30–50 μM) none of the lipid soluble spin probes tested was cytotoxic. At 100 μM only 5-Doxylstearate inhibited cell survival, whereas 12-Doxylstearate and 16-Doxylstearate had no effect.

Internal motions in myosin. 2.

The high-resolution 1H NMR detected internal motions in myosin and myosin subfragment 1 (S1) [Highsmith, S., Akasaka, K., Konrad, M., Goody, R., Holmes, K., Wade-Jardetzky, N., & Jardetzky, O. (1979) Biochemistry 18, 4238--4244] were unperturbed by induced changes in the rate of protein tumbling, and the mobile regions proved inaccessible to added surface-directed paramagnetic probes. The rate of tumbling was changed by changing the solvent viscosity for S1 or by aggregation to thick filaments for myosin. Neither manipulation caused a measurable broadening of the narrow lines in the spectrum. Sulfhydryl-directed covalently attached nitroxide spin-labels, soluble nitroxide spin-labels, and MnCl2 were used to probe the surface. Unique labeling at the fastest reacting thiol of S1 had no effect on the NMR spectrum. Multiple labeling of thiols caused a small but detectable broadening of the narrow peaks. Soluble spin-labels and MnCl2 had a very small effect on the narrow bands even in great excess. The results substantiate the notion that myosin has internal motions that are independent of the overall rate of rotation and suggest that the mobile structure is mainly in the interior of the S1 moiety. This supports a model in which actin quenches the internal motions of myosin by changing the structure of myosin upon binding.

Paramagnetic fluorescence quenching in chlorophyll a containing vesicles: Evidence for the localization of chlorophyll

Spinprobes (fatty acids, androstane, cholestane) that differ in the location of the paramagnetic centre relative to the polar membrane surface have been incorporated into lipid vesicles containing chlorophyll a. Quenching of the Chl a fluorescence is observed following the Stern-Vollmer-relationship. Quenching is more effective with spinprobes carrying the nitroxide group near the polar head groups of the lipid moiety. Quenching is also observed using a water soluble spinlabel. The porphyrin ring of the Chl a molecules is suggested to be localized in the polar head group region of the bilayer membrane.

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 compliment.

Changes in the restriction of molecular rotational diffusion of water-soluble spin labels during fatty acid starvation of yeast

Yeast mutants lacking fatty acid synthetase activity (fas-) die when deprived of saturated fatty acid under conditions which are otherwise growth-supporting. The spin label technique is used to show that restriction of molecular rotational diffusion of spin label molecules dissolved in aqueous zones increases several fold under conditions of fatty acid starvation while the apparent physical state of cellular hydrocarbon zones remains essentially unchanged. We focus attention on the cellular aqueous interior as the potential site of alteration under selective starvation conditions. Correspondences exist between restriction of molecular motion of water soluble spin labels dissolved in the cell and loss of cell viability. The correspondences to changes in the molecular motion of hydrocarbon soluble spin labels are much less or are not detectable.

A spin-label study of the viscosity profile of sarcoplasmic reticular vesicles

Sarcoplasmic reticular vesicles were prepared from both lobster and rabbit muscle. A variety of water-soluble, lipid-soluble, and alkylating spin labels were used to treat the sarcoplasmic reticular vesicles. All spin label analyses were carried out with and without NiCl 2. Nickel is used to remove spin label signal originating from outside of, on the surface of, or localized in the outermost part of the outer bilayer half of the sarcoplasmic reticular membrane. We conclude that the hydrocarbon portion of sarcoplasmic reticular vesicles has symmetry in regard to the physical properties that limit spin label motion; however, we find that the membrane interface limits spin label motion more on the inner surface than the outer surface and that the trapped aqueous volume which is sampled by water soluble spin labels inside the vesicle enclosure limits spin label motion much more than the average aqueous medium outside.