Microscopic versus macroscopic diffusion in model membranes by electron spin resonance spectral-spatial imaging.

The macroscopic and the microscopic diffusion coefficients of a phospholipid spin label (16-PC) in the model membrane 1-palmitoyl-2-oleoyl-sn-glycero-phosphatidylcholine have been measured simultaneously in the same sample utilizing the new technique of spectral-spatial electron spin resonance imaging. The macroscopic diffusion coefficient Dmacro for self-diffusion of 16-PC spin label is obtained from imaging the concentration profiles as a function of time, and it is (2.3 +/- 0.4) x 10(-8) cm2/s at 22 degrees C. The microscopic diffusion coefficient Dmicro for relative diffusion of the spin probes is obtained from the variation of the spectral line broadening with spin label concentration, which is due to spin-spin interactions. Dmicro is found to be substantially greater than Dmacro for the same sample at the same conditions, and is estimated to be at least (1.0 +/- 0.4) x 10(-7) cm2/s. Possible sources for their difference are briefly discussed in terms of the models used for Dmicro.     

Interaction of transmembrane helices in ATP synthase subunit a in solution as revealed by spin label difference NMR

Subunit a in the membrane traversing F0 sector of Escherichia coli ATP synthase is known to fold with five transmembrane helices (TMHs) with residue 218 in TMH IV packing close to residue 248 in TMH V. In this study, we have introduced a spin label probe at Cys residues substituted at positions 222 or 223 and measured the effects on the Trp epsilon NH indole NMR signals of the seven Trp residues in the protein. The protein was purified and NMR experiments were carried out in a chloroform-methanol-H2O (4:4:1) solvent mixture. The spin label at positions 222 or 223 proved to broaden the signals of W231, W232, W235 and W241 located at the periplasmic ends of TMH IV and TMH V and the connecting loop between these helices. The broadening of W241 would require that the loop residues fold back on themselves in a hairpin-like structure much like it is predicted to fold in the native membrane. Placement of the spin label probe at several other positions also proved to have broadening effects on some of these Trp residues and provided additional constraints on folding of TMH IV and TMH V. The effects of the 223 probes on backbone amide resonances of subunit a were also measured by an HNCO experiment and the results are consistent with the two helices folding back on themselves in this solvent mixture. When Cys and Trp were substituted at residues 206 and 254 at the cytoplasmic ends of TMHs IV and V respectively, the W254 resonance was not broadened by the spin label at position 206. We conclude that the helices fold back on themselves in this solvent system and then pack at an angle such that the cytoplasmic ends of the polypeptide backbone are significantly displaced from each other.     

Interaction of transmembrane helices in ATP synthase subunit a in solution as revealed by spin label difference NMR

Subunit a in the membrane traversing F₀ sector of Escherichia coli ATP synthase is known to fold with five transmembrane helices (TMHs) with residue 218 in TMH IV packing close to residue 248 in TMH V. In this study, we have introduced a spin label probe at Cys residues substituted at positions 222 or 223 and measured the effects on the Trp ?NH indole NMR signals of the seven Trp residues in the protein. The protein was purified and NMR experiments were carried out in a chloroform-methanol-H₂O (4:4:1) solvent mixture. The spin label at positions 222 or 223 proved to broaden the signals of W231, W232, W235 and W241 located at the periplasmic ends of TMH IV and TMH V and the connecting loop between these helices. The broadening of W241 would require that the loop residues fold back on themselves in a hairpin-like structure much like it is predicted to fold in the native membrane. Placement of the spin label probe at several other positions also proved to have broadening effects on some of these Trp residues and provided additional constraints on folding of TMH IV and TMH V. The effects of the 223 probes on backbone amide resonances of subunit a were also measured by an HNCO experiment and the results are consistent with the two helices folding back on themselves in this solvent mixture. When Cys and Trp were substituted at residues 206 and 254 at the cytoplasmic ends of TMHs IV and V respectively, the W254 resonance was not broadened by the spin label at position 206. We conclude that the helices fold back on themselves in this solvent system and then pack at an angle such that the cytoplasmic ends of the polypeptide backbone are significantly displaced from each other.     

Trehalose effect on low temperature protein dynamics: fluctuation and relaxation phenomena

We performed spectral diffusion experiments in trehalose-enriched glycerol/buffer-glass on horseradish peroxidase where the heme was replaced by metal-free mesoporphyrin IX, and compared them with the respective behavior in a pure glycerol/buffer-glass (Schlichter et al., J. Chem. Phys. 2000, 112:3045-3050). Trehalose has a significant influence: spectral diffusion broadening speeds up compared to the trehalose-free glass. This speeding up is attributed to a shortening of the correlation time of the frequency fluctuations most probably by preventing water molecules from leaving the protein interior. Superimposed to the frequency fluctuation dynamics is a relaxation dynamics that manifests itself as an aging process in the spectral diffusion broadening. Although the trehalose environment speeds up the fluctuations, it does not have any influence on the relaxation. Both relaxation and fluctuations are governed by power laws in time. The respective exponents do not seem to change with the protein environment. From the spectral dynamics, the mean square displacement in conformation space can be determined. It is governed by anomalous diffusion. The associated frequency correlation time is incredibly long, demonstrating that proteins at low temperatures are truly nonergodic systems.     

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.     

Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation recovery and continuous wave power saturation electron paramagnetic resonance.

Melittin spin-labeled specifically with a nitroxide at positions 7, 21, 23, or the amino terminus was bound to phospholipid membranes, and the exposure of the spin label to the aqueous phase was investigated by measurement of Heisenberg exchange with chromium oxalate in the solution. The exchange frequency was determined by saturation recovery electron paramagnetic resonance (EPR) using a loop-gap resonator. This method allows use of very low concentrations (less than 1 mM) of chromium oxalate compared with conventional measurements of EPR line broadening (typically 50 mM), thus avoiding problems associated with high metal ion concentration. Differences in exchange frequency between the various positions were also estimated by continuous wave power saturation methods. In either approach, the spin label at lysine 7 was found to be the most exposed to chromium oxalate whereas that at lysine 23 was found to be the least exposed. This is consistent with a model for the membrane bound peptide in which an amphiphilic helix lies with its axis parallel to the bilayer surface and the hydrophobic moment points toward the bilayer interior.     

A spin label method for measuring internal volumes in liposomes or cells, applied to Ca-dependent fusion of negatively charged vesicles

A new spin label - broadening agent system for measuring trapped volumes of vesicles or cells is described. The method seems to be more advantageous than existing procedures when volumes of highly negatively charged vesicles are to be determined. The membrane permeable spin label is TEMPONE (2,2,6,6-tetramethyl piperidone-N-oxyl), and the nonpermeable broadening agent is chromium oxalate (K3Cr(C2O4)3). Absolute values for the trapped volumes down to 0.1% in 0.1 ml can be measured with an accuracy of about +/- (1-10%). The method is used to study the final volume of fused phosphatidylserine vesicles as a function of the temperature at which the Ca-induced fusion takes place.     

High pressure EPR studies of protein mobility in reversed micelles

We have investigated the effect of pressure on structural properties of subtilisin solubilized in reversed micelles of Tween-85/isopropanol in hexane. Electron paramagnetic resonance (EPR) spectra of spin-labeled enzyme indicate a reduction in spin-label mobility when the enzyme is transferred from aqueous solution to the microemulsion. One explanation for the spectral broadening is a change in the protein's active-site conformation and/or dynamics. However, over a W(0) range of 80 to 180, EPR spectroscopy could detect no change in the enzyme's environment, conformation, or molecular dynamics. The EPR spectra also contained a contribution from free spin label located in an environment with a polarity roughly between that of propanol and bulk water. No changes in the polarity surrounding the free spin label nor in the enzyme's structural properties were evident at pressures up to 10,000 psi. Previous work has demonstrated that pressure can be used to manipulate the size of some reversed micelles, and the EPR data indicated that for this system such pressure tuning of micellar properties will not adversely affect the structure of solubilized enzyme. (c) 1994 John Wiley & Sons, Inc.     

Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes: I. Effect of temperature on membrane protein and lipid mobility

A protein spin label and lipid spin probes were used to study the temperature-dependent motion of protein and lipid, respectively, in barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes. Using membranes from seedlings grown at 20°C, the temperature-dependence of the relative motion of membrane surface spin probes and a spin label covalently attached to membrane proteins suggested abrupt changes in the lipid and protein mobilities at about 12°C. Spin probe spin-spin exchange broadening and fluorescent probe eximer formation indicated apparent temperature-induced alterations in probe lateral diffusion within the membrane at about 12 to 14°C. The results suggest the presence of temperature-induced quasicrystalline lipid clusters which may influence the activity of membrane-bound enzymes.     

Chromium oxalate: a new spin label broadening agent for use with thylakoids.

Potassium tris(oxalato)chromate(III) trihydrate (chromium ixalate) has been shown to be a more useful broadening agent than potassium ferricyanide for the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amine (Tempamine) in thylakoid suspensions. Our data show that chromium oxalate is less permeable than ferricyanide, does not inhibit thylakoid electron transport or photophosphorylation, and is not photoreduced by thylakoids.     

Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.     

Chromium oxalate: A new spin label broadening agent for use with thylakoids

Potassium tris(oxalato)chromate(III) trihydrate (chromium oxalate) has been shown to be a more useful broadening agent than potassium ferricyanide for the spin label 2,2,6,6-tetramethylpiperidine-N-oxy-4-amine (Tempamine) in thylakoid suspensions. Our data show that chromium oxalate is less permeable than ferricyanide, does not inhibit thylakoid electron transport or photophosphorylation, and is not photoreduced by thylakoids.     

High field/high frequency saturation transfer electron paramagnetic resonance spectroscopy: increased sensitivity to very slow rotational motions

Saturation transfer electron paramagnetic resonance (ST-EPR) spectroscopy has been employed to characterize the very slow microsecond to millisecond rotational dynamics of a wide range of nitroxide spin-labeled proteins and other macromolecules in the past three decades. The vast majority of this previous work has been carried out on spectrometers that operate at X-band ( approximately 9 GHz) microwave frequency with a few investigations reported at Q-band ( approximately 34 GHz). EPR spectrometers that operate in the 94-250-GHz range and that are capable of making conventional linear EPR measurements on small aqueous samples have now been developed. This work addresses potential advantages of utilizing these same high frequencies for ST-EPR studies that seek to quantitatively analyze the very slow rotational dynamics of spin-labeled macromolecules. For example, the uniaxial rotational diffusion (URD) model has been shown to be particularly applicable to the study of the rotational dynamics of integral membrane proteins. Computational algorithms have been employed to define the sensitivity of ST-EPR signals at 94, 140, and 250 GHz to the correlation time for URD, to the amplitude of constrained URD, and to the orientation of the spin label relative to the URD axis. The calculations presented in this work demonstrate that these higher microwave frequencies provide substantial increases in sensitivity to the correlation time for URD, to small constraints in URD, and to the geometry of the spin label relative to the URD axis as compared with measurements made at X-band. Moreover, the calculations at these higher frequencies indicate sensitivity to rotational motions in the 1-100-ms time window, particularly at 250 GHz, thereby extending the slow motion limit for ST-EPR by two orders of magnitude relative to X- and Q-bands.     

Inositol-less death in yeast results in a simultaneous increase in intracellular viscosity.

Inositol auxotrophs of yeast developing on isositol-deficient medium continue protein synthesis for 4-6 h, lose viability rapidly after 6 h, and show an increase in cytoplasmic viscosity as measured by spin label rotational motion. Cycloheximide prevents the rapid loss of cell viability, stops protein synthesis, and simultaneously prevents an increase in cytoplasmic viscosity. From these observations, we infer that intracellular translational diffusion is upset as a consequence of inositol starvation. Cell death may be caused by a modified intracellular diffusion environment.     

Librational motion of an "immobilized" spin label: hemoglobin spin labeled by a maleimide derivative.

The spin label Tempo-maleimide, when "immobilized" in hemoglobin, is shown to exhibit motional fluctuation whose amplitude and/or frequency depend on temperature and solution conditions. These motional fluctuations are observable by several electron spin resonance techniques. For desalted hemoglobin the fluctuations are detectable at approximately -15 degrees C using saturation transfer techniques and at approximately +25 degrees C using line-width measurements of normal absorption spectra. In ammonium sulfate precipitated hemoglobin, however, motional fluctuations are not detectable by either technique up to at least 40 degrees C. The most probable mechanism for spin-label motion appears to be either fluctuations in protein conformation which affect the label binding site or conformational transitions of the nitroxide ring itself. These motional fluctuations are shown to introduce a librational character to the overall label motion during hemoglobin rotational diffusion, with the librational motion significantly affecting the use of spin-label spectral shapes to calculate hemoglobin rotational correlation times.     

EPR studies of spin-labeled bovine plasma amine oxidase: the nature of the substrate-binding site.

The carbonyl cofactor of bovine plasma amine oxidase (EC 1.4.3.6), recently shown to be 6-hydroxydopa (also known as topa), has been spin labeled to the extent of one label per enzyme dimer molecule, using 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) and 4-hydrazino-TEMPO followed by reduction with borohydride. By studying the EPR spectra of the labeled enzyme, it has been deduced that there is no magnetic interaction between the copper and the spin label, and that the spin label is at least 1.3 nm distant from the copper(II) ion in the resting enzyme. The bound label is strongly immobilized, is in a sterically constricted environment, and is not accessible to small anions. Removal of the copper does not alter the EPR spectrum of the label. The results are similar to results for porcine plasma amine oxidase, and show that the copper is not close to, and does not directly interact with, the topa-bound substrate.     

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.     

Spin-label studies of the pH-induced random coil to α-helix conformational transition in poly(L-lysine)

Poly(L -lysine) of various molecular weights between 2700 and 475,000 was spin-labeled. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined by calculation of the rotational correlation time as the poly(L -lysine) underwent the pH-induced random coil to α-helix conformational transition. In general, the rotational correlation time of the nitroxide increased as the pH was increased, indicating a more restricted environment for the spin label when poly(L -lysine) is deprotonated. For the high-molecular-weight poly(L -lysine) this corresponds to the formation of the α-helix and indicates that the side chain–side chain interaction and decreased segmental motion of the backbone (slightly) restricts the motion of the spin label. For the 2700-molecular-weight poly(L -lysine), previously shown not to assume a helical conformation at high pH, the increase in the rotational correlation time of the spin label indicates that the side chain–side chain interaction takes place after deprotonation but without helix formation. This may indicate that helix formation per se is not needed to produce the observed effect even with the high-molecular-weight polymers. The rotational correlation time of the spin label at a particular pH did not depend on the molecular weight of the poly(L -lysine) over the 200-fold range of molecular weights. This indicates that the rotational correlation time reflects the rotational mobility of the spin label in a localized environment and not the rotational diffusion of the entire macromolecule.     

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-dodecylphosphopiperidine, have been incorporated into dodecylphosphocholine 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.     

Nitroxide-labeled guanine as an ESR spin probe for structural study of DNA

A guanine derivative with a covalently linked nitroxide spin label has been devised. The spin label was incorporated into oligodeoxynucleotides by post-synthetic modification. The local environment of a variety of G-rich DNA is detectable by ESR using this spin probe.