Transfer Electron Paramagnetic Resonance Spectroscopy Research Articles

2 mm waveband saturation transfer electron paramagnetic resonance of conducting polymers

The 2 mm waveband (140 GHz) saturation transfer electron paramagnetic resonance (ST-EPR) spectroscopy has been employed to characterize the very slow microsecond to millisecond librational macromolecular dynamics of a wide range of conducting polymers. It is possible at this waveband to determine separately spin relaxation and dynamics affecting ST-EPR spectra. Higher microwave frequency provides substantial increases sensitivity of the method to the anisotropic macromolecular motion in conducting polymers and broadens the interval of correlation times up to 1-80 ms, thereby extending the slow-motion limit for ST-EPR by two orders of magnitude compared with convenient wavebands EPR.

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 (∼9 GHz) microwave frequency with a few investigations reported at Q-band (∼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.

Effect of adenosine 5'-[beta,gamma-imido]triphosphate on myosin head domain movements.

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) was used to study the orientation of probe molecules in muscle fibers in different intermediate states of the ATP hydrolysis cycle. A separate procedure was used to obtain ST EPR spectra with precise phase settings even in the case of samples with low spectral intensity. Fibers prepared from rabbit psoas muscle were labeled with isothiocyanate spin labels at the reactive thiol sites of the catalytic domain of myosin. In comparison with rigor, a significant difference was detected in the orientation-dependence of spin labels in the ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[CH2]P) states, indicating changes in the internal dynamics and domain orientation of myosin. In the AdoPP[CH2]P state, approximately half of the myosin heads reflected the motional state of ADP-myosin, and the other half showed a different dynamic state with greater mobility.

The regulatory domain of the myosin head behaves as a rigid lever.

The regulatory domain of the myosin head is believed to serve as a lever arm that amplifies force generated in the catalytic domain and transmits this strain to the thick filament. The lever arm itself either can be passive or may have a more active role storing some of the energy created by hydrolysis of ATP. A structural correlate which might distinguish between these two possibilities (a passive or an active role) is the stiffness of the domain in question. To this effect we have examined the motion of the proximal (ELC) and distal (RLC) subdomains of the regulatory domain in reconstituted myosin filaments. Each subdomain was labeled with a spin label at a unique cysteine residue, Cys-136 of ELC or Cys-154 of mutant RLC, and its mobility was determined using saturation transfer electron paramagnetic resonance spectroscopy. The mobility of the two domains was similar; the effective correlation time (tau(eff)) for ELC was 17 micros and that for RLC was 22 micros. Additionally, following a 2-fold change of the global dynamics of the myosin head, effected by decreasing the interactions with the filament surface (or the other myosin head), the coupling of the intradomain dynamics remained unchanged. These data suggest that the regulatory domain of the myosin head acts as a single mechanically rigid body, consistent with the regulatory domain serving as a passive lever.

Molecular mobility in the cytoplasm of lettuce radicles correlates with longevity

AbstractMolecular mobility is hypothesised to be a key factor influencing storage stability of seeds because it may control the rate of deteriorative reactions responsible for reduced shelf life. The relationship between the longevity of lettuce seeds and the molecular mobility was investigated in the cytoplasm of lettuce radicles. Longevity of lettuce seeds was predicted using the viability equation of Ellis and Roberts, and the molecular mobility was determined by saturation transfer electron paramagnetic resonance spectroscopy measurements of rotational motion of a polar spin probe inserted into the cytoplasm. Increasing the temperature resulted in faster rotational motion and shorter longevity. There was a linear relationship between the logarithms of rotational motion and estimated seed longevity for temperatures above 5°C. Below 5°C, there was a deviation from linearity. Based on the hypothesis that the molecular mobility in the cytoplasm determines the rate of detrimental reactions, seed longevity at sub-zero temperatures was predicted by extrapolation of the rotational motion using the linear relationship. Predictions of longevity at sub-zero temperatures based on rotational motion indicated longer survival times than those based on the viability equation. A kinetic approach to ageing using molecular mobility measurements is expected to improve our understanding of seed storage stability and might eventually lead to realistic predictions of longevity at low temperatures.

Is There a Role for Oligosaccharides in Seed Longevity? An Assessment of Intracellular Glass Stability

We examined whether oligosaccharides extend seed longevity by increasing the intracellular glass stability. For that purpose, we used a spin probe technique to measure the molecular mobility and glass transition temperature of the cytoplasm of impatiens (Impatiens walleriana) and bell pepper (Capsicum annuum) seeds that were osmo-primed to change oligosaccharide content and longevity. Using saturation transfer electron paramagnetic resonance spectroscopy, we found that the rotational correlation time of the polar spin probe 3-carboxy-proxyl in the cytoplasm decreased, together with longevity, as a function of increasing seed water content, suggesting that longevity may indeed be regulated by cytoplasmic mobility. Osmo-priming of the seeds resulted in considerable decreases in longevity and oligosaccharide content, while the sucrose content increased. No difference in the glass transition temperature was found between control and primed impatiens seeds at the same temperature and water content. Similarly, there was no difference in the rotational motion of the spin probe in the cytoplasm between control and primed impatiens and bell pepper seeds. We therefore conclude that oligosaccharides in seeds do not affect the stability of the intracellular glassy state, and that the reduced longevity after priming is not the result of increased molecular mobility in the cytoplasm.

Molecular mobility in the cytoplasm: an approach to describe and predict lifespan of dry germplasm.

Molecular mobility is increasingly considered a key factor influencing storage stability of biomolecular substances, because it is thought to control the rate of detrimental reactions responsible for reducing the shelf life of, for instance, pharmaceuticals, food, and germplasm. We investigated the relationship between aging rates of germplasm and the rotational motion of a polar spin probe in the cytoplasm under different storage conditions using saturation transfer electron paramagnetic resonance spectroscopy. Rotational motion of the spin probe in the cytoplasm of seed and pollen of various plant species changed as a function of moisture content and temperature in a manner similar to aging rates or longevity. A linear relationship was established between the logarithms of rotational motion and aging rates or longevity. This linearity suggests that detrimental aging rates are associated with molecular mobility in the cytoplasm. By measuring the rotational correlation times at low temperatures at which experimental determination of longevity is practically impossible, this linearity enabled us to predict vigor loss or longevity. At subzero temperatures, moisture contents for maximum life span were predicted to be higher than those hitherto used in genebanks, urging for a reexamination of seed storage protocols.

Functional and structural differences in skeletal and cardiac myosins. A molecular dynamic approach

Conventional and saturation transfer electron paramagnetic resonance spectroscopy and differential scanning calorimetry were used to study the internal dynamics and stability of cardiac myosin. Intact and LC 2-deficient myosin isolated from bovine heart were spin-labelled with maleimide and iodoacetamide probe molecules at the SH1 sites. It was found that the probe molecules rotate with an effective rotational correlation time of 42 ns, which is at least six times shorter than the rotational correlation time of the same label on skeletal myosin. Addition of MgADP induces intrinsic changes in the multisubunit structure of myosin, but it does not lead to changes of the overall rotational properties of the myosin head. Temperature dependence of the EPR spectra of maleimide-labelled myosin shows continuous decrease of the spectral parameters (intensity ratio of the peak heights, hyperfine splitting) at increasing temperature. However, marked changes were obtained at about 16°C in LC 2-deficient myosin. DSC measurements also support the view that the removal of the LC 2 light chain produces change in the internal structure of cardiac myosin.

Dynamic aspects of the incorporation of proteins into biological membranes.

The contributions of intramembranous and extramembranous segments of transmembrane proteins to frictional forces have been studied by covalently attached 14N- and 15N-indane dione and maleimide spin labels using saturation transfer electron spin resonance spectroscopy. The role of molecular size and membrane viscosity is discussed in determining rotational mobilities of proteins. By comparing the measured rotational correlation times with the predictions of hydrodynamic models the aggregation states of transmembrane proteins is estimated. On increasing the viscosity of the aqueous phase by polyols the viscous drag of the extramembranous segments of proteins is increased and from systematic hydrodynamic measurements the size of the protruding segments can be estimated. The role of slowed molecular diffusion is briefly discussed in the inhibition of enzymatic activity.

Internal flexibility of cardiac myosins

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) and differential scanning calorimetry (DSC) were used to study the motional dynamics and segmental flexibility of cardiac myosins. Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with isothiocyanate- or maleimide-based probe molecules at the reactive sulfhydryl sites (Cys-697 and Cys-707) of the motor domain. The maleimide probe molecules attached to human cardiac myosin rotated with an effective rotational correlation time of 33 ns which was at least eight times shorter than the rotational correlation time of the same label on skeletal myosin (260 ns). In the presence of MgADP and MgADP plus orthovanadate, flexibility changes in the multisubunit structure of myosins were detected, but this did not lead to changes of the overall rotational property of the myosin heads. Significant difference in the internal flexibility was detected on myosin samples isolated from ischemic tissue, the rotational correlation time decreased to 25 ns. DSC measurements supported the view that addition of nucleotides produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that there is an intersite communication between the nucleotide binding domain and the 20 kDa subunit where the reactive thiol sites are located.

Effect of the C-terminal proline repeats on ordered packing of squid rhodopsin and its mobility in membranes

Negative stain electron microscopy and saturation transfer electron spin resonance spectroscopy have been used to compare the lattice ordering and in-plane membrane mobility of full-length and C-terminally cleaved squid rhodopsin. The C-terminus of squid rhodopsin contains a negatively charged region followed by 9–10 repeats of a proline-rich sequence, not found in rhodopsins other than those of cephalopod invertebrates, but similar proline repeats are found in other, unrelated membrane proteins. We find that the proline repeats cluster the rhodopsins into small groups, interfering with two-dimensional crystallization and maintaining their mobility in the membrane.

Trans-unsaturated lipid dynamics: modulation of dielaidoylphosphatidylcholine acyl chain motion by ethanol

Acyl chain dynamics of the trans-unsaturated lipid, dielaidoylphosphatidylcholine (DEPC), were studied by conventional and saturation transfer electron paramagnetic resonance spectroscopy of aqueous dispersions of DEPC spin labeled with lecithins having doxyl groups at positions 5, 10, and 14 on the sn-2 chain. The gel to liquid crystalline transition is concerted with simultaneous increases in rotational motion about the long axis of the acyl chain (libration) and in gauche-trans conformational interconversions (wobble). Relative to saturated lecithins at similar reduced temperatures the double bond (a) slowed libration by an order of magnitude in both phases, while wobble motions were several times slower, and (b)-produced a pronounced stiffness of the acyl chain near the double bond. Ethanol (0-1.6 M), in addition to its well-known colligative effect on the phase transition, was found to decrease the bilayer order in a concentration-dependent manner. This effect was smaller in the gel than in the liquid crystalline phase, most pronounced next to the double bond, and weakest deep in the bilayer. Ethanol affected slow motions little in the gel phase but wobble and libration correlation times were markedly decreased in the liquid crystalline phase.

Maleimide, Iodoacetamide, Indanedione, and Chloromercuric Spin Label Reagents with Derivatized Nitroxide Rings as ESR Reporter Groups for Protein Conformation and Dynamics

The syntheses of eight nitroxide spin labels which bear maleimide, iodoacetamide, indanedione, or chloromercuric reactive groups and, in addition, a second substituent in the nitroxide ring are presented. The second substituent groups range from hydrophobic and hydrophilic esters to carboxylic acid and secondary and tertiary amine groups. The resulting spin labels are characterized with respect both to protein covalent modification and to the electron spin resonance spectral properties of the bound labels. The effect of the various substituents in the spin label on the reactivity toward the membrane-bound shark rectal gland and pig kidney Na,K-ATPase is described. The spectral differences between immobilized and mobile groups observed by electron spin resonance for the different protein-bound spin labels show that, by selecting an appropriate derivative for modification, a large range of different motional sensitivities of the reporter group can be obtained. Such different series of spin labels should therefore be useful for detecting mobility changes arising from conformational transitions in proteins by conventional electron spin resonance spectroscopy or for measurement of protein rotational diffusion using saturation transfer electron spin resonance spectroscopy. The chloromercuric series is found to be particularly useful because of the high reactivity, the lack of reversibility that potentially is associated with the Michael addition reaction, and the wide range of rotational mobility that is exhibited by the different derivatives.

Structure-function relationships in the inorganic salt-induced precipitation of α-chymotrypsin

alpha-Chymotrypsin (alpha CT) was used as a model protein to study the effects of salt-induced precipitation on protein conformation. Process parameters investigated included the type and amount of salt used to induce precipitation. The salts studied included Na2SO4, NaCl, NaBr, KBr and KSCN. Precipitate secondary structure content was examined via laser Raman spectroscopy. Conventional and saturation transfer electron paramagnetic resonance spectroscopy were employed to probe the tertiary structure of the active site in spin-labelled alpha CT precipitates. As the molal surface tension increment of the inducing salt increased, the beta-sheet content increased and the alpha-helix content decreased. There was no significant variation in secondary structure with the amount of salt used. The fraction of precipitate that recovered activity on redissolution was correlated with the change in secondary structure content. Spin-labelled precipitate spectra indicated that the active site remains unaltered during precipitation. Molecular modelling was employed to investigate how physical property of alpha CT were affected by these types of conformational change. Estimated physical property changes could not account entirely for observed deviations from current equilibrium theory for salt-induced precipitation. The spectroscopic observations were also combined with activity/solubility results to propose a mechanism for the salt-induced precipitation of globular proteins.

Analysis of saturation transfer electron paramagnetic resonance spectra in terms of amplitude and phase

A systematic analysis of saturation transfer (ST) electron paramagnetic resonance spectroscopy was developed to separate an observed absorption signal either into an amplitude and a phase spectrum or into an in-phase and an ST spectrum. We established a phase reference procedure, replacing the low-power phase null method by a high-power phase maximum method. This is an extension of the finding that a peak-to-peak height and a single integration of the second harmonic absorption display vary sinusoidally as functions of the modulation phase [Y. Shimoyama and H. Watari, Appl. Spectrosc. 39, 170 (1985)]. Based on the sinusoidal variation in the peak-to-peak height, we found a phase shift at each magnetic field and could evaluate the amplitude and phase of a signal vector. The sinusoidal variation of the single integrated value allowed the absorption signal to be separated into an in-phase and an ST spectrum. The high-power phase maximum method provides a remedy of the phase setting problem in ST spectroscopy by which the in-phase signal can be detected at any microwave field.

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude ( τ c ( L″ L ) ~ 5 × 10 −5 and 8 × 10 −5 s , respectively, at 2 °C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.

Segmental flexibility and head-head interaction in scallop myosin: A Study using saturation transfer electron paramagnetic resonance spectroscopy

Saturation transfer electron paramagnetic resonance spectroscopy was used to investigate the rotational motion of the head domains of native and desensitized scallop myosin and its proteolytic subfragments. Scallop myosin was spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl, which reacted with a heavy chain residue in the subfragment 1 domain. As previously shown for rabbit skeletal muscle myosin (Thomas et al., 1975). the two head domains of native scallop myosin appear to have independent motion (rotational correlation time, τ, = 0.8 × 10 −7s for subfragment 1: 1.4 × 10 −7s for myosin). However, removal of a regulatory light chain, to effect desensitization of the actin-activated ATPase, was associated with an increase in τ for myosin to a value of 2.4 × 10 −6s. The Ca 2+ sensitivity and initial correlation time were restored on recombination of the regulatory light chain in the presence of Mg 2+ Sedimentation velocity profiles in an analytical ultracentrifuge indicated that the desensitized myosin preparations were largely monomeric and therefore the change in τ appears to reflect an intramolecular event. Addition of EDTA to spin-labelled scallop heavy meromyosin caused an immediate 2.5 to 4-fold increase in τ and a partial desensitization of the ATPase activity. Comparable experiments with subfragment 1 yielded a barely detectable increase in τ (1.5-fold) in the first ten minutes. The restricted rotational motion observed in desensitized myosin and heavy meromyosin could arise by a conformational change in the subfragment 1 subfragment 2 hinge region or by an association of one head with its partner. The latter mechanism, involving the exposed light chain binding site, would also explain the preferential release of one regulatory light chain from scallop myosin, and might account for some other co-operative effects observed in this molecule (Bagshaw. 1980).

Rotational diffusion of cytochrome P-450 in the microsomal membrane—Evidence for a clusterlike organization from saturation transfer electron paramagnetic resonance spectroscopy

Rotational diffusion of cytochrome P-450 in rabbit liver microsomes has been studied by saturation transfer EPR spectroscopy. Sulfhydryl groups of cytochrome P-450 were selectively modified using a maleimide spin label. The effective rotational correlation time for the rotation of cytochrome P-450 was calculated to be about 480 μ s which corresponds to a very strong immobilization thus evidencing protein aggregation within the membrane. The anisotropic character of the spectra indicates a nonspherical shape and/or anisotropic rotational motion of the cluster. The temperature dependence of the rotational correlation time shows a relatively sharp break at about 4 °C but only small changes above this temperature. The break at about 4 °C is probably caused by the onset of the cluster rotation. Below 4 °C the enzyme is almost immobilized. A similar complete immobilization can be achieved by treating the microsomes with glutaraldehyde.

Phase Dependence of Saturation Transfer EPR Signals and Estimated Rotational Correlation Times

The second harmonic absorption out-of-phase signal (V2′ mode) is usually observed in saturation transfer electron paramagnetic resonance spectroscopy to determine slow rotational correlation times. It is known that adjustment of 90° out-of-phase is critically important to detect accurate ST-EPR spectra. The digitized ST-EPR spectrometer using the Fourier transformation method developed by the authors has enabled determination of the phase dependence of ST-EPR signals simply by calculating V2′ signals under the condition of various phase deviations from exactly 90° out-of-phase. The overall 90° out-of-phase accuracy of the present system was confirmed to be within 0.3°. Phase dependence of both the ST-EPR parameters and the correlation times estimated from them was quantitatively discussed. Conclusively, accuracy of phase-adjustment is required to be within ±0.5° for less than 10%, and ±2° or 3° for less than approximately 30% errors on the correlation times. For determination of correlation times the parameter L″/L provides better accuracy than the parameter C′/C, even if 90° out-of-phase is set with some phase deviation.

35-GHz (Q-band) saturation transfer electron paramagnetic resonance studies of rotational diffusion.

The extension of saturation transfer electron paramagnetic resonance spectroscopy (ST-EPR) to an observational frequency of 35 GHz (Q band) is described. At this frequency the spectral resolution is greatly enhanced over that afforded at the 9.5-GHz (X-band) frequency used in most of the ST-EPR studies published to date. Thus, Q-band operation may provide an approach for the detailed analysis of the slow anisotropic motions believed to occur in many biomolecular systems. The spectral characteristics and the effects of various instrumental settings are described in detail for a model system of spin-labeled hemoglobin in water-glycerol solutions. Several spectral parameters are defined, and their potential use in monitoring various types of anisotropic motion is considered.