Cation-Induced Changes in the Membrane Fluidity of Isolated Corn Mitochondria as Determined by Nitroxide Spin Labels

Cooke, A., Collison, D, Mabbs, F. E. and Earnshaw, M. J. 1985.Cation-induced changes in the membrane fluidity of isolatedcorn mitochondria as determined by nitroxide spin labels.—J.exp Bot. 36: 1799–1808. The addition of Ca^2– or La³⁺ to non-energized corn mitochondria,with incorporated spin labels, results in an increase in 2T_uof the membrane surface label I (12, 3) and an increase in ofthe membrane core label 1(1, 14). These results indicate a decreasein the motion of the label within the mitochondrial membranes. Decreasing the temperature also increases the 2T_u and torque;of I (12, 3)- and I (1, 14)-labelled corn mitochondria respectively.This suggests that a fall in temperature acts similarly to theaddition of cations in that the freedom of motion of spin labelsin the membrane is limited. Comparing the temperature-inducedchanges in label motion to those of Ca²⁺ implies that the membranecore is more sensitive to Ca²⁺ -induced changes in motion thanis the surface. A survey of a range of multivalent cations suggests that theireffect on spin label motion is largely non-specific and probablydue to cation binding. Key words: Calcium, mitochondria, membranes, fluidity     

Interaction of nitroxide spin labels with chloroplasts

Chloroplasts isolated from oats eliminated the electron spin resonance (ESR) signals from spin labels in white light and partially restored them in far-red light. Only the white light-mediated reaction was blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In contrast, oat (Avena sativa L. cv. Garry and Park) leaf mesophyll protoplasts oxidized the spin labels in both white and far-red light, with and without DCMU. Light had no obvious effect on spin label motion within chloroplast membranes. The results suggest that, in isolated chloroplasts, nitroxide spin labels may be reduced by photosystem I within the thylakoid bilayer resulting in loss of the ESR signals. The reduced forms may be reoxidized by an element of the photosynthetic electron transport chain which operates between the DCMU block and the photosystem I reaction center. In addition, a light-mediated destruction of the spin labels occurs in both chloroplasts and protoplasts. The reduced form of the nitroxide (i.e. the hydroxylamine) may be resistant to this destruction.     

Location and orientation relative to the micelle surface for glucagon in mixed micelles with dodecylphosphocholine EPR and NMR studies

The spin labels, 5-doxylstearate, 12-doxylstearate, 16-doxylstearate and 1-oxyl-2,2,6,6-tetramethyl-4-dodecylphospiperidine, have been incorporated into dodecylphospocholine micelles and mixed dodecylphosphocholine/ glucagon micelles. The EPR spectral parameters for the different spin labels and the ¹H- and ¹³C-NMR relaxation rates for nuclei of the detergent molecules indicated that inclusion of up to one spin label molecule per micelle had little influence on the spatial organization of the micelles. Furthermore, the location and environment of the spin labels in the dodecylphosphocholine micelles were not noticeably affected by the addition of glucagon and the ¹H-NMR spectra observed for glucagon in mixed spin label/deuterated dodecylphosphocholine/glucagon micelles showed that the different spin labels had essentially no effect on the conformation of glucagon. Approximate spatial locations within the micelle for the nitroxide moieties of the different spin labels were determined from the NMR relaxation rates observed for different nuclei of dodecylphosphocholine. On this basis, the line broadening of individually assigned glucagon ¹H-NMR lines by the different spin labels was used to determine the approximate orientation of the polypeptide chain with respect to the micelle surface. Overall, the data indicate that the glucagon backbone runs roughly parallel to the micelle surface, with the depth of immersion adjusted so that polar and apolar side chains can be oriented towards the surface or interior of the micelle, respectively.     

Spin-label techniques for monitoring macromolecular rotational motion: empirical calibration under nonideal conditions.

Practical techniques are demonstrated for determining rotational correlation times of macromolecules from the first harmonic absorption electron spin reasonance spectra of tightly bound spin labels. The techniques are developed to compensate for such nonideal conditions as residual label motion, temperature dependence of rigid limit spectral parameters, and the presence of inhomogeneous line broadening. These effects are all shown to be of importance in monitoring the rotational motion of carbonmonoxyhemoglobin which is spin labeled with the tightly bound nitroxide label, 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxy. Spin-label interactions with other paramagnetic agents are also shown to produce spectral changes which are qualitatively similar to, but quantitatively different from, those resulting from increases in the rate of rotational motion.     

Use of double spin-labeled histones to monitor histone-chromatin integration

Tyrosine-specific nitroxide spin labels have been synthesized that utilize either deuterium or deuterium and [15N] isotopic substitution within the nitroxide ring. These probes have been used to differentially spin label and simultaneously monitor both histones H1 and H5, during the displacement of endogenous spin-labeled H1 from reconstituted chromatin by exogenously added spin-labeled H5.     

Spin Label Translational Diffusion in Solid Tristearin

Translational diffusion of the intermediate chain length spin label 7N14 has been detected and studied in a lipid environment which is in the bulk solid state. Under favorable circumstances this can occur at temperatures as much as 50°C below the optical melting point. Translational diffusion allows 7N14 molecules to coalesce into impurity pools of high spin label concentration. Two other spin labels, 2N3 and 14N27, do not show a tendency to form such impurity pools. While 2N3 undergoes rapid tumbling at temperatures far below the melting point of the tristearin matrix, the molecules remain in an isolated state with no evidence of spin exchange. 14N27 is restricted in rotational motion in the solid matrix and also does not form impurity pools.     

The conformations of nitroxide-labelled fatty acid probes of membrane structure as studied by 2H-NMR

The electron spin resonance (ESR) spectrum of a nitroxide spin probe intercalated in a membrane is influenced by the amplitude of anisotropic motion of the nitroxide group and by the geometry of the oxazolidine ring of the nitroxide. In the analysis of the ESR spectra of nitroxide-labelled fatty acid probes, it is generally assumed that the five-membered oxazolidine ring system is oriented rigidly perpendicular to the long molecular axis of the probe. This assumption is tested in the present study, using ²H-NMR of specifically deuterium-labelled nitroxide spin probes. Evidence is presented that the nitroxide does not display the assumed geometry in membranes. The departure from this geometry depends on the position of the nitroxide label on the acyl chain, with a more pronounced departure for position 5 relative to position 12. These and previous data provide an explanation for the discrepancies between spin-probe ESR and ²H-NMR order parameters in membranes.     

Theoretical analysis of the molecular motion of spin labels in membranes. ESR spectra of labeled Bacillus subtilis membranes

Electron spin resonance (ESR) spectral line shapes are calculated for a nitroxide radical undergoing rapid motion within a cone. The analysis is correct to second order, and explicit expression are derived for the hyperfine splittings and g-values by averaging both the secular and pseudosecular terms within the Hamiltonian. The simulated spectra are found to closely resemble those observed experimentally over a wide range of temperatures for stearic acid spin labels in cytoplasmic membranes of Bacillus subtilis. The present approach offers a simple, yet realistic way of interpreting spectra of nitroxide spin labels such as fatty acids and steroids when the motion is anisotropic.     

Behavior of spin labels in a variety of interdigitated lipid bilayers

The behavior of a number of spin labels in several lipid bilayers, shown by X-ray diffraction to be interdigitated, has been compared in order to evaluate the ability of the spin label technique to detect and diagnose the structure of lipid bilayers. The main difference between interdigitated and non-interdigitated gel phase bilayers which can be exploited for determination of their structure using spin labels, is that the former have a much less steep fluidity gradient. Thus long chain spin labels with the nitroxide group near the terminal methyl of the chain, such as 16-doxylstearic acid, its methyl ester, or a phosphatidylglycerol spin label containing 16-doxylstearic acid (PG-SL), are more motionally restricted and/or ordered in the interdigitated bilayer than in the non-interdigitated bilayer. This difference is large enough to be of diagnostic value for all three spin labels in the interdigitated bilayers of dihexadecylphosphatidylcholine, dipalmitoylphosphatidylcholine/ethanol, and 1,3-dipalmitoylphosphatidylcholine. However, it is not large enough to be of diagnostic value at low temperatures. Use of probes with the nitroxide group closer to the apolar/polar interface reveals that these latter interdigitated bilayers are more disordered or less closely packed. As the temperature is increased, however, the motion of the PG-SL does not increase as much in these interdigitated bilayers as in non-interdigitated bilayers. The difference in the motion and/or order of PG-SL between interdigitated and non-interdigitated bilayers is large enough at higher temperatures to be of value in diagnosing the structure of the bilayers. Thus by choice of a suitable spin label and a suitable temperature, this technique should prove useful for detection and diagnosis of lipid bilayer structure with a good degree of reliability. Caution must, of course be exercised, as with any spectroscopic technique. Spin labels will also be invaluable for more detailed studies of known interdigitated bilayers, which would be time- and material-consuming, if carried out using X-ray diffraction solely.     

Structure of the lipid phase of rauscher murine leukemia virus

The lipid-containing membrane of Rauscher murine leukemia virus was studied using stearic acid spin labels with the nitroxide ring on the C₅ and C₁₆ positions. The environment of the C₅ spin label was found to be much more rigid than that of the C₁₆ spin label. This result, which parallels similar observations in red cell membranes and influenza virus, suggests that the lipid phase of Rauscher murine leukemia virus is arranged in a bilayer.     

Acridine spin labels as probes for nucleic acids.

Adridine spin labels, 4-[9-(6-chloro-2-methoxy)-acridylamino]- 2,2,6,6-tetramethyl-1-piperidinyloxy (I) and 4-(9-acridylamino)- 2,2,6,6-tetramethyl-1-piperidinyloxy (II), have been synthesized and their interaction with nucleic acids studied by means of electron spin resonance (ESR). The ESR spectra of labels I and II in the presence of calf thymus DNA were characteristic of highly immobilized nitroxide radicals with maximum hyperfine splittings (2T_ˌˌ) of 58.7 and 55.5 G, respectively. The melting temperature (T_m) of DNA, determined in the presence of labels I and II by the ESR technique, were closely similar to those obtained by spectrophotometric methods. The ESR spectrum of label I bound to calf liver RNA and yeast RNA indicated that the nitroxide group of this label was highly mobile. These results suggest that spin labels I and II are suitable noncovalent probes for nucleic acids.     

Membrane hydrocarbon thickness modulates the dynamics of a membrane transport protein

Nitroxide spin labels were incorporated into selected sites within the β-barrel of the bacterial outer-membrane transport protein BtuB by site-directed mutagenesis, followed by chemical modification with a methanethiosufonate spin label. The electron paramagnetic resonance lineshapes of the spin-labeled side chain (R1) from these sites are highly variable, and have spectral parameters that reflect secondary structure and local steric constraints. In addition, these lineshape parameters correlate with crystallographic structure factors for Cα carbons, suggesting that the motion of the spin label is modulated by both the local modes of motion of the spin label and the local dynamics of the protein backbone. Experiments performed as a function of lipid composition and sample temperature indicate that nitroxide spin labels on the exterior surface of BtuB, which face the membrane hydrocarbon, are not strongly influenced by the phase state of the bulk lipids. However, these spectra are modulated by membrane hydrocarbon thickness. Specifically, the values of the scaled mobility parameter for the R1 lineshapes are inversely proportional to the hydrocarbon thickness. These data suggest that protein dynamics and structure in BtuB are directly coupled to membrane hydrophobic thickness.     

Saturation transfer electron paramagnetic resonance on membrane bound proteins. II-Absence of rotational diffusion of the cholinergic receptor protein in Torpedo marmorata membrane fragments.

We have applied the technique of saturation transfer electron paramagnetic resonance to study the rotational diffusion of spin labeled membrane bound cholinergic receptors from Torpedo marmorata. Two different spin labels were used: a spin labeled maleimide derivative which binds covalently to proteins and a long chain spin labeled acylcholine which binds reversibly with a high affinity to the receptor protein. The maleimide spin label has a motion whose rotational correlation time is τ₂ > 10^?3 sec. The long chain spin labeled acylcholine indicates slightly more motion ($\text{τ}{}_2\text{? 10}{}^{\mathrm{?4}}\text{sec}$), but the nitroxide in this latter case is probably more loosely bound.     

A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data

The measurement of (1)H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed α-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed α-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.     

Monitoring RNA base structure and dynamics using site-directed spin labeling

Site-directed spin labeling utilizes site-specific attachment of a stable nitroxide radical to probe the structure and dynamics of macromolecules. In the present study, a 4-thiouridine base is introduced at each of six different positions in a 23-nucleotide RNA molecule. The 4-thiouridine derivatives were subsequently modified with one of three methanethiosulfonate nitroxide reagents to introduce a spin label at specific sites. The electron paramagnetic resonance spectra of the labeled RNAs were analyzed in terms of nitroxide motion and the RNA solution structure. At a base-paired site in the RNA helix, where the nitroxide has weak or no local interactions, motion of the nitroxide is apparently dominated by rotation about bonds within the probe. The motion is similar to that found for a structurally related probe on helical sites in proteins, suggesting a similar mode of motion. At other sites that are hydrogen bonded and stacked within the helix, local interactions within the RNA molecule modulate the nitroxide motion in a manner consistent with expectations based on the known structure. For a base that is not structurally constrained, the mobility is higher than at any other site, presumably due to motion of the base itself. These results demonstrate the general utility of the 4-thiouridine/methanethiosulfonate coupling method to introduce nitroxide spin labels into RNA and the ability of the resulting label to probe local structure and dynamics.     

High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.     

Ionic strength-dependent alterations of membrane structure of red blood cells

Electron paramagnetic resonance (EPR) measurements using various fatty acid spin labels were performed on membranes of intact human erythrocytes at physiological, and at low ionic strength. In the case of spin probes bearing the nitroxide near the polar head group, a less restricted motion at low ionic strength was seen than with those labels with a nitroxide deeper within the hydrophobic tail of the membrane. Although these data clearly show an influence of ionic strength on membrane structure, and possibly a modified protein-lipid interaction, they cannot be simply discussed in terms of an altered membrane fluidity.     

Saturated and unsaturated lipid spin labels with terminally located nitroxide groups

Synthetic routes are described to a new series of nitroxide lipid spin labels useful for studying the effects of unsaturation and chain length on motion experienced by nitroxide spin labels in biological membrane systems. The labels incorporate a terminally-located proxyl nitroxide group on a saturated or unsaturated fatty acid chain. Syntheses utilize as the key step either an alkylation of an acetylide anion with a nitroxide iodide or else a Wittig coupling between a nitroxide ylid and an aldehyde. Spin labels described include 17-proxylstearolic acid, 17-peroxyl-stearic acid, 17-proxyloleic acid, 16-proxylheptadecanoic acid, 9-proxyldecanoic acid and two phosphatidyl choline derivatives.     

Synthesis of charged amphipathic nitroxide lipid spin labels and an example of their application in membrane studies

The synthesis of a series of amphipathic nitroxide lipid spin labels is reported. Thus, 12-proxylhexadecanol has been converted into the versatile fatty acid spin label 14-proxylstearic acid. This substance was used to prepare 14-proxylstearyltrimethylammonium methanesulfonate, a positively charged label, and 14-proxylstearylmethyl phosphate sodium salt, a negatively charged label. Also prepared in the doxyl series were quaternary ammonium salts derived from 16-doxyl- and 7-doxylstearic acid. The positively charged and negatively charged proxyl labels were used in a preliminary experiment to investigate the role of charge in their interaction with reconstituted cytochrome oxidase. The average binding affinity of the negatively charged label is approximately 2-fold higher than that of the positively charged label at pH 7.4. At pH 5.5 the average relative affinity for negatively charged label is about 3.5-fold higher than that of positively charged label, suggesting that the ionizable group(s) on the protein can interact with the lipid headgroup.     

Two dimensional diffusion of small molecules on protein surfaces: an EPR study of the restricted translational diffusion of protein-bound spin labels

Heisenberg spin exchange rates and dipole-dipole spin lattice relaxation rates for deuterated ¹⁴N- and ¹⁵N-spin labels bound selectively to the histidine His15 and to the lysines Lys13, 96, 97 of the lysozyme molecule have been determined with the aid of electron spin resonance spectroscopy. The results can be interpreted in terms of a two dimensional translational diffusion of the nitroxide tips of the spin labels along the protein surface within restricted surface areas. The spin labels are regarded as models for long amino acid side chains and as probes for the dynamics of protein and water in the vicinity of the protein surface. The translational diffusion coefficient DP_II is reduced by a factor of between six and thirty compared to the value of D found for the spin labels in bulk water, its value for T = 295 K is given by (1.3±0.6)·10^–10m²s^–1 D (2.4±0.3) 10^–11 m²s^–1. Offprint requests to: H.-J. Steinhoff