Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 A. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.     

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.     

Calculation of electron paramagnetic resonance spectra from Brownian dynamics trajectories: application to nitroxide side chains in proteins.

We present a method to simulate electron paramagnetic resonance spectra of spin-labeled proteins that explicitly includes the protein structure in the vicinity of the attached spin label. The method is applied to a spin-labeled polyleucine alpha-helix trimer. From short (6 ns) stochastic dynamics simulations of this trimer, an effective potential energy function is calculated. Interaction with secondary and tertiary structures determine the reorientational motion of the spin label side chains. After reduction to a single particle problem, long stochastic dynamic trajectories (700 ns) of the spin label side-chain reorientation are calculated from which the Lamor frequency trajectory and subsequently the electron paramagnetic resonance spectrum is determined. The simulated spectra agree well with experimental electron paramagnetic resonance spectra of bacteriorhodopsin mutants with spin labels in similar secondary and tertiary environments as in the polyleucine.     

Lipid Librations at the Interface with the Na,K-ATPase

Transitions between conformational substates of membrane proteins can be driven by torsional librations in the protein that may be coupled to librational fluctuations of the lipid chains. Here, librational motion of spin-labeled lipid chains in membranous Na,K-ATPase is investigated by spin-echo electron paramagnetic resonance. Lipids at the protein interface are targeted by using negatively charged spin-labeled fatty acids that display selectivity of interaction with the Na,K-ATPase. Echo-detected electron paramagnetic resonance spectra from native membranes are corrected for the contribution from the bilayer regions of the membrane by using spectra from dispersions of the extracted membrane lipids. Lipid librations at the protein interface have a flat profile with chain position, whereas librational fluctuations of the bilayer lipids increase pronouncedly from C-9 onward, then flatten off toward the terminal methyl end of the chains. This difference is accounted for by increased torsional amplitude at the chain ends in bilayers, while the amplitude remains restricted throughout the chain at the protein interface with a limited lengthening in correlation time. The temperature dependence of chain librations at the protein interface strongly resembles that of the spin-labeled protein side chains, suggesting solvent-mediated transitions in the protein are driven by fluctuations in the lipid environment.     

Spin-label studies of lipid-protein interactions with reconstituted band 3, the human erythrocyte chloride-bicarbonate exchanger.

Lipid-protein interactions in reconstituted band 3 preparations were investigated by using spin-labeled lipids in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Purified erythrocyte band 3 was reconstituted into egg phosphatidylcholine liposomes at high protein density with preservation predominantly of the dimeric state. Lipid-protein associations were revealed by the presence of a component in the EPR spectra that, when compared to spectra obtained from protein-free bilayers, indicated that lipid chain motions are restricted by interactions with the protein. From the fraction of the motionally restricted component obtained from the phosphatidylcholine spin-label, a value of 64 +/- 14 annular lipids per band 3 dimer was obtained. This agrees with a value of 62 for the number of lipids that may be accommodated around the electron density map of a band 3 dimer. Selectivity of various spin-labeled lipids for the protein revealed that androstanol had a lower affinity for the band 3 interface, whereas a distinct preference was observed for the negatively charged lipids phosphatidylglycerol and stearic acid over phosphatidylcholine. This preference for negatively charged lipids could not be screened by 1-M salt, indicating that electrostatic lipid-protein interactions are not dominant. Estimates of annular lipid exchange rates from measured acyl chain segmental motions suggested that the rate of exchange between bilayer and boundary lipids was approximately 10(6) s(-1), at least an order of magnitude slower than the rate of lipid lateral diffusion in protein-free bilayers.     

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.     

Increased Sensitivity and Extended Range of Distance Measurements in Spin-Labeled Membrane Proteins: Q-Band Double Electron-Electron Resonance and Nanoscale Bilayers

We report a significant methodological advance in the application of double electron-electron resonance (DEER) spectroscopy to measure long-range distances in spin-labeled membrane proteins. In the pseudo two-dimensional environment of proteoliposomes, a steep intermolecular background shapes DEER signals leading to long accumulation times, complicating data analysis and reducing the maximal measurable distances from 70 Å down to ∼40-50 Å. To eliminate these limitations, we took advantage of the homogeneity and monodispersity of a class of discoidal nanoscale phospholipid bilayers in conjunction with the micromolar DEER sensitivity at Q-band (34 GHz) microwave frequency. Spin-labeled mutants of the ABC transporter MsbA were functionally reconstituted at a ratio of one functional dimer per nanoscale apolipoprotein-bound bilayer (NABB). DEER echo intensities from NABB-reconstituted MsbA have linear baselines reflecting a three-dimensional spatial distribution. This results in an order-of-magnitude higher sensitivity at Q-band relative to proteoliposomes and restores the maximal observable distance effectively increasing experimental throughput. The advances described here set the stage for the use of DEER spectroscopy to analyze conformational dynamics of sample-limited eukaryotic membrane proteins.     

Insect crossbridges, relaxed by spin-labeled nucleotide, show well-ordered 90 degrees state by X-ray diffraction and electron microscopy, but spectra of electron paramagnetic resonance probes report disorder.

The structure of glycerinated Lethocerus insect flight muscle fibers, relaxed by spin-labeled ATP and vanadate (Vi), was examined using X-ray diffraction, electron microscopy and electron paramagnetic resonance (e.p.r.) spectra. We obtained excellent relaxation of MgATP quality as determined by mechanical criteria, using vanadate trapping of 2' spin-labeled 3' deoxyATP at 3 degree C. In rigor fibers, when the diphosphate analog is bound in the absence of Vi, the probes on myosin heads are well-ordered, in agreement with electron microscopic and X-ray patterns showing that myosin heads are ordered when attached strongly to actin. In relaxed muscle, however, e.p.r. spectra report orientational disorder of bound (Vi-trapped) spin-labeled nucleotide, while electron microscopic and X-ray patterns both show well-ordered bridges at a uniform 90 degrees angle to the filament axis. The spin-labeled nucleotide orientation is highly disordered, but not completely isotropic; the slight anisotropy observed in probe spectra is consistent with a shift of approximately 10% of probes from angles close to 0 degrees to angles close to 90 degrees. Measurements of probe mobility suggest that the interaction between probe and protein remains as tight in relaxed fibers as in rigor, and thus that the disorder in relaxed fibers arises from disorders of (or within) the protein and not from disorder of the probe relative to the protein. Fixation of the relaxed fibers with glutaraldehyde did not alter any aspect of the spectrum of the Vi-trapped analog, including the slight order observed, showing that the extensive inter- and intra-molecular cross-linking of the first step of sample preparation for electron microscopy had not altered relaxed crossbridge orientations. Two models that may reconcile the apparently disparate results obtained on relaxed fibers are presented: (1) a rigid myosin head could possess considerable disorder in the regular array about the thick filament; or (2) the nucleotide site could be on a disordered, probably distal, domain of myosin, while a more proximal region is well ordered on the thick filament backbone. Our findings suggest that when e.p.r. probes signal disorder of a local site or domain, this is complementary, not contradictory, to signals of general order. The e.p.r. spectra show that a portion of the myosin molecule can be disordered at the same time as the X-ray diffraction and electron microscopy show the bulk of myosin head mass to be uniformly oriented and regularly arrayed.     

Association of methanol and ethanol with heme proteins.

The behavior of ferrihemoglobin and ferrimyoglobin in widely varying concentrations of the lowest four alcohols has been studied by optical and electron paramagnetic resonance absorption spectroscopy. Methanol and ethanol, at concentrations too low to cause general conformational destabilization of the protein, produce both optical and electron paramagnetic resonance absorption spectral changes in ferrihemoglobin. These changes arise from equilibrium associations, characterized by dissociation constants at 25 degrees C of about 40 and 200 mM, respectively, for the methanol-ferrihemoglobin and ethanol-ferrihemoglobin complexes so formed. Other optical spectral changes appear when the methanol concentration exceeds 3.5 M and the ethanol, 1.0 M. At concentrations lower than 0.5 M, 1- and 2-propanol produce spectral changes of this second kind. At room temperature no optical evidence has been found that the propanols associate with ferrihemoglobin in the manner of methanol and ethanol. Methanol and ethanol at low concentration have specific effects, characterized by electron paramagnetic resonance spectral differences, upon ferric alphaSH chains. All four alcohols, over a wide range of concentrations, reduce the symmetry of electron paramagnetic resonance spectra from frozen solutions of ferrihemoglobin; even at the high end of this concentration range, none of the alcohols reduces the symmetry of electron paramagnetic resonance spectra from frozen ferrimyoglobin. Ferrimyoglobin and catalase association with methanol is measurable optically; the binding is about five and sixty times weaker, respectively, for these two proteins as compared with ferrihemoglobin.     

Synthesis and properties of novel porphyrin spin probes containing isoindoline nitroxides

We report the synthesis of novel spin-labeled porphyrins containing isoindoline nitroxides (TMIO-APTPP and TMIO-APTSPP) and their manganese complexes (Mn-TMIO-APTPP and Mn-TMIO-APTSPP). These compounds represent potential new tools for electron paramagnetic resonance (EPR) as well as novel spin probes. Both TMIO-APTPP and TMIO-APTSPP have characteristic UV absorption peaks of free base porphyrin, while the characteristic absorption peaks of their manganese complexes Mn-TMIO-APTPP and Mn-TMIO-APTSPP shifted to shorter wavelengths. Electron paramagnetic resonance (EPR) spectroscopy indicated that these compounds all exhibit hyperfine splittings characteristic of EPR spectra of tetramethylisoindoline nitroxides, the typical nitroxide g values of approximately 2.006, and nitrogen isotropic hyperfine coupling constants (a(N) values) of about 14 G (293 K). The observed linewidths (La) for TMIO-APTSPP (0.73 G) and Mn-TMIO-APTSPP (0.65 G) in distilled water are significantly narrower than for TMIO-APTPP (1.475 G) and Mn-TMIO-APTPP (1.55 G) in chloroform.     

Determination of the orientation of T4 lysozyme vectorially bound to a planar-supported lipid bilayer using site-directed spin labeling

Site-directed spin labeling is used to investigate the structure of adsorbed T4 lysozyme (T4L). A monolayer of T4L is prepared by tethering the protein selectively via a His-tag to the chelating headgroups (NTA Ni) of a planar quartz-supported lipid bilayer. This results in a vectorially oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance spectra. Similar measurements of spin-labeled lipid bilayers were used to characterize the structure and dynamics of the supports. Electron paramagnetic resonance line shape was analyzed using the stochastic Liouville equation approach developed by Freed and co-workers. The simulations reveal a conservation of the secondary and tertiary structure of T4L upon adsorption although slight conformational changes in the presence of the surface can be detected by probing tertiary contact sites. The orientation of the entire protein was deduced on the basis of an anisotropic motional model for the spin-labeled side chain. In addition, a polar order but azimuthal disorder of the molecules was assumed to fit the data. These results demonstrate the utility of site-directed spin labeling in combination with spectral simulation to study not only the secondary and tertiary structure of adsorbed proteins in monolayer coverage but also their orientation with respect to the surface.     

Synthesis and properties of spin-labeled angiotensin derivatives

We have synthesized three derivatives of the peptide hormone angiotensin, containing as their N-terminal residue the spin-labeled amino acid 2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylicacid. (TOAC). The angiotensin analogs displayed considerable biological activity, indicating that the spin label is not too great a perturbation for the hormone-receptor interaction. Studies of the effect of pH upon the electron paramagnetic resonance (EPR) spectra of the spin-labeled angiotensins indicated that deprotonation of the terminal amino group leads to changes in spectral parameters similar to those displayed by model compounds (TOAC and TOAC-glycine). In view of the slow exchange between the two forms at pH values where both the protonated and unprotonated forms coexist in considerable amounts, computer simulations demonstrate that the EPR spectra are superpositions of the spectra for each form. The EPR spectra of the spin-labeled hormone derivatives were shown to be indicative of a pH-dependent conformationai change, corroborating previous conclusions drawn from other studies. This study demonstrates the usefulness of spin-labeled analogs for the investigation of conformational properties of small peptides.     

Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.     

Large-scale rotational motions of proteins detected by electron paramagnetic resonance and fluorescence.

Direct spectroscopic measurements of rotational motions of proteins and large protein segments are crucial to understanding the molecular dynamics of protein function. Fluorescent probes and spin labels attached to proteins have proved to be powerful tools in the study of large-scale protein motions. Fluorescence depolarization and conventional electron paramagnetic resonance (EPR) are applicable to the study of rotational motions in the nanosecond-to-microsecond time range, and have been used to demonstrate segmental flexibility in an antibody and in myosin. Very slow rotational motions, occurring in the microsecond-to-millisecond time range, are particularly important in supramolecular assemblies, where protein motions are restricted by association with other molecules. Saturation transfer spectroscopy (ST-EPR), a recently developed electron paramagnetic resonance (EPR) technique that permits the detection of rotational correlation times as long as 1 ms, has been used to detect large-scale rotational motions of spin-labeled proteins in muscle filaments and in membranes, providing valuable insights into energy transduction mechanisms in these assemblies.     

Integrated analysis of the conformation of a protein-linked spin label by crystallography,EPR and NMR spectroscopy

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein–protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.     

Synthesis and micellar formation of spin-labeled lysolecithin.

A spin-labeled lysolecithin, 1-[12'-(N-oxyl-4",4"-dimethyloxazolidine)-stearoyl]-sn-glycero-3-phosphorylcholine, has been synthesized in which the spin is covalently attached to its fatty acyl chain. The electron spin resonance spectra of this lysolecithin is an aqueous solution generally showed sharp three resonance lines superposed on a broad resonance line. Investigation of changes in the signal intensity of these spectra against the concentration of lysolecithin led to the inference that the sharp lines are due to monomers of lysolecithin while the broad one to micelles. The critical micellar concentration was consistent with that evaluated from the spectral shift of a dye. In the electron spin resonance spectra obtained from spin-labeled lysolecithin solutions with various amounts of dimyristoyllecithin, the line width of broad signal arised from micellar spin-labeled lysolecithin broadened as increase of dimyristoyllecithin. The line-broadening thus observed was briefly discussed.     

Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients

New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinson's disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.     

Probes of membrane electrostatics: synthesis and voltage-dependent partitioning of negative hydrophobic ion spin labels in lipid vesicles.

Two spin-labeled derivatives of the hydrophobic anion trinitrophenol have been synthesized and characterized in lipid vesicles. In the presence of lipid vesicles, the electron paramagnetic resonance (EPR) spectra of these probes are a composite of both membrane-bound and aqueous populations; as a result, the membrane-aqueous partitioning can be determined from their electron paramagnetic resonance spectra. The effect of transmembrane potentials on the membrane-aqueous partitioning of these spin-labeled hydrophobic ions was examined in phosphatidylcholine vesicles formed by extrusion. Inside positive membrane potentials promote an increase in the binding of these probes that is quantitatively accounted for by a simple thermodynamic model used previously to describe the partitioning of paramagnetic phosphonium ions. The transmembrane migration rates of these ions are dependent on the dipole potential, indicating that these ions transit the membrane in a charged form. The partitioning of the probe is also sensitive to the membrane surface potential, and this dependence is accurately accounted for using the Gouy-Chapman Stern formalism. As a result of the membrane dipole potential, these probes exhibit a stronger binding and a more rapid transmembrane migration rate compared with positive hydrophobic ion spin labels and provide a new set of negatively charged hydrophobic ion probes to investigate membrane electrostatics.     

Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model.

Algorithms have been developed for the calculation of saturation transfer electron paramagnetic resonance (ST-EPR) spectra of a nitroxide spin-label assuming uniaxial rotational diffusion, a model that is frequently used to describe the global rotational dynamics of large integral membrane proteins. One algorithm explicitly includes terms describing Zeeman overmodulation effects, whereas the second more rapid algorithm treats these effects approximately using modified electron spin-lattice and spin-spin relaxation times. Simulations are presented to demonstrate the sensitivity of X-band ST-EPR spectra to the rate of uniaxial rotational diffusion and the orientation of the nitroxide probe with respect to the diffusion axis. Results obtained by using the algorithms presented, which are based on the transition-rate formalism, are in close agreement with those obtained by using an eigenfunction expansion approach. The effects of various approximations used in the simulation algorithms are considered in detail. Optimizing the transition-rate formalism to model uniaxial rotational diffusion results in over an order of magnitude reduction in computation time while allowing treatment of nonaxial A- and g-tensors. The algorithms presented here are used to perform nonlinear least-squares analyses of ST-EPR spectra of the anion exchange protein of the human erythrocyte membrane, band 3, which has been affinity spin-labeled with a recently developed dihydrostilbene disulfonate derivative, [15N,2H13]-SL-H2DADS-MAL. These results suggest that all copies of band 3 present in intact erythrocytes undergo rotational diffusion about the membrane normal axis at a rate consistent with a band 3 dimer.     

Saturation transfer EPR spectroscopy on spin-labeled muscle fibers using a loop-gap resonator.

Previously, saturation transfer (ST-EPR) studies of biomolecular dynamics have involved the use of a resonant cavity and the V'2 display (absorption, second harmonic, out of phase). In the present study, we replaced the resonant cavity with a loop-gap resonator and used the U'1 display (dispersion, first harmonic, out of phase) to study spin-labeled muscle fibers. The new resonator and display showed several advantages over those previously used. It produced virtually noiseless U'1 spectra on a 0.4 microliter sample using a 4 min scan; previous U'1 experiments on spin-labeled muscle, using a conventional rectangular cavity, resulted in an unacceptably low signal-to-noise ratio. The high filling factor of the resonator facilitated the study of these extremely small fiber bundles and permitted high microwave field intensities to be achieved at much lower incident microwave power levels, thus greatly enhancing the signal-to-noise ratio in U'1 experiments. This reduction in the noise level made it possible to benefit from the other advantages of U'1 over V'2, such as stronger signals, simpler line shapes, and simpler data analysis. For these muscle fiber samples, the resulting sensitivity (signal/noise/sample volume) of the U'1 signals was greater than 100 times that of V'2 signals obtained in a conventional cavity. Another advantage of the U'1 display is that signals from weakly immobilized probes, i.e., probes that have nanosecond rotational mobility relative to the labeled protein (myosin), are greatly suppressed relative to strongly immobilized probes. This reduces the ambiguity of spectral analysis, and eliminates the need for chemical treatments [e.g., using K3Fe(CN)6] that were previously required in muscle fibers and other systems. Further suppression of this weakly immobilized component was achieved in U'1 spectra by increasing the microwave power and decreasing the field modulation frequency.