Carbon-centered radicals in isolated soy proteins.

Solid-state electron paramagnetic resonance (EPR) spectroscopy of commercial samples of isolated soy proteins (ISP) revealed a symmetrical free-radical signal typical of carbon-centered radicals (g= 2.005) ranging from 2.96 x 10(14) to 6.42 x 10(14) spins/g. The level of free radicals in ISP was 14 times greater than similar radicals in sodium caseinate, 29 times greater than egg albumin, and about 100 times greater levels than casein. Nine soy protein powdered drink mixes contained similar types of free radicals up to 4.10 x 10(15) spins/g of drink mix, or up to 6.4 times greater than the highest free-radical content found in commercial ISP. ISP samples prepared in the laboratory contained trapped radicals similar to the levels in commercial ISP samples. When ISP was hydrated in 2.3 mM sodium erythorbate or 8.3 mM L-cysteine, frozen and dried, the level of trapped free radicals increased by about 17- and 19-fold, respectively. The ESR spectrum of defatted soybean flakes contained overlapping signals from the primary free-radical peak (g= 2.005) and a sextet pattern typical of manganese-II. The manganese signal was reduced in the laboratory ISP and very weak in the commercial ISP.

The sensitivity of saturation transfer electron paramagnetic resonance spectra to restricted amplitude uniaxial rotational diffusion.

Computational methods have been developed to model the effects of constrained or restricted amplitude uniaxial rotational diffusion (URD) on saturation transfer electron paramagnetic resonance (ST-EPR) signals observed from nitroxide spin labels. These methods, which have been developed to model the global rotational motion of intrinsic membrane proteins that can interact with the cytoskeleton or other peripheral proteins, are an extension of previous work that described computationally efficient algorithms for calculating ST-EPR spectra for unconstrained URD (Hustedt and Beth, 1995, Biophys. J. 69:1409-1423). Calculations are presented that demonstrate the dependence of the ST-EPR signal (V'(2)) on the width (Delta) of a square-well potential as a function of the microwave frequency, the correlation time for URD, and the orientation of the spin-label with respect to the URD axis. At a correlation time of 10 micros, the V'(2) signal is very sensitive to Delta in the range from 0 to 60 degrees, marginally sensitive from 60 degrees to 90 degrees, and insensitive beyond 90 degrees. Sensitivity to Delta depends on the correlation time for URD with higher sensitivity to large values of Delta at the shorter correlation times, on the microwave frequency, and on the orientation of the spin-label relative to the URD axis. The computational algorithm has been incorporated into a global nonlinear least-squares analysis approach, based upon the Marquardt-Levenberg method (Blackman et al., 2001, Biophys. J. 81:3363-3376). This has permitted determination of the correlation time for URD and the width of the square-well potential by automated fitting of experimental ST-EPR data sets obtained from a spin-labeled membrane protein and provided a new automated method for analysis of data obtained from any system that exhibits restricted amplitude URD.

Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes.

The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702-4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32 degrees full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of approximately 73 degrees full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.

Nitroxide spin-spin interactions: applications to protein structure and dynamics.

Measurement of the distance between two spin label probes in proteins permits the spatial orientation of elements of defined secondary structure. By using site-directed spin labeling, it is possible to determine multiple distance constraints and thereby build tertiary and quaternary structural models as well as measure the kinetics of structural changes. New analytical methods for determining interprobe distances and relative orientations for uniquely oriented spin labels have been developed using global analysis of multifrequency electron paramagnetic resonance data. New methods have also been developed for determining interprobe distances for randomly oriented spin labels. These methods are being applied to a wide range of structural problems, including peptides, soluble proteins, and membrane proteins, that are not readily characterized by other structural techniques.

Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data.

For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar splittings can be observed in continuous wave electron paramagnetic resonance (CW-EPR) spectra for interelectron distances as large as 30 A using perdeuterated probes. In this work, algorithms are developed for calculating CW-EPR spectra of immobilized, dipolar coupled nitroxides, and then used to define the limits of sensitivity to the interelectron distance as a function of geometry and microwave frequency. Secondly, the CW-EPR spectra of N epsilon-spin-labeled coenzyme NAD+ bound to microcrystalline, tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, and 94 GHz. These data have been analyzed, using a combination of simulated annealing and global analysis, to obtain a unique fit to the data. The values of the intermitroxide distance and the five angles defining the relative orientation of the two nitroxides are in reasonable agreement with a molecular model built from the known crystal structure. Finally, the effect of rigid body isotropic rotational diffusion on the CW-EPR spectra of dipolar coupled nitroxides has been investigated using an algorithm based on Brownian dynamics trajectories. These calculations demonstrate the sensitivity of CW-EPR spectra to dipolar coupling in the presence of rigid body rotational diffusion.

Increased rotational mobility and extractability of band 3 from protein 4.2-deficient erythrocyte membranes: evidence of a role for protein 4.2 in strengthening the band 3-cytoskeleton linkage.

Band 3 (anion-exchange protein 1-[AE1]) is the major integral membrane protein of human erythrocytes and links the membrane to the underlying cytoskeleton via high-affinity binding to ankyrin. It is unclear whether other cytoskeletal proteins participate in strengthening the ankyrin-band 3 linkage, but a putative role for protein 4.2 (P4.2) has been proposed based on the increased osmotic fragility and spherocytic morphology of P4.2-deficient red blood cells (RBCs). The present study was designed to investigate the hypothesis that P4.2 has a direct role in strengthening the band 3-cytoskeleton linkage in human RBCs, by measuring independent features of this interaction in normal and P4.2-deficient RBCs. The features examined were the rotational mobility of band 3 assayed by time-resolved phosphorescence emission anisotropy (TPA), and the extractability of band 3 by octyl-beta-glucoside, the latter being a nonionic detergent that selectively extracts only band 3 that is not anchored to the cytoskeleton. We find that the amplitude of the most rapidly rotating population of band 3 (correlation time, approximately 30 to 60 microseconds) is increased 81% and 67% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts. The amplitude of the intermediate speed rotating population of band 3 (correlation time, approximately 200 to 500 microseconds) is increased 23% and 8% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts, at the expense of the slowly rotating component (correlation time, approximately 2,000 to 3,000 microseconds, amplitude decreased 43% and 39% in P4.2NIPPON and band 3MONTEFIORE, respectively) and immobile component (immobile on this experimental time scale; amplitude decreased 26% and 10% in P4.2NIPPON and band 3MONTEFIORE, respectively) of band 3. These results demonstrate that P4.2 deficiency partially removes band 3 rotational constraints, ie, it increases band 3 rotational mobility. The nonionic detergent octyl-beta-glucoside, which does not disturb band 3-cytoskeleton associations, ie, it extracts only band 3 that is not attached to the cytoskeleton, extracted 30% and 61% more band 3 from P4.2NIPPON and band 3MONTEFIORE ghost membranes, respectively, compared with control ghosts. The octyl-beta-glucoside ghost extracts from both P4.2-deficient phenotypes were enriched in band 3 oligomeric species (tetramers, higher-order oligomers, and aggregates) compared with controls. Since band 3 oligomers selectively associate with the cytoskeleton, these results are consistent with a weakened band 3-cytoskeleton linkage in P4.2-deficient RBC membranes. P4.2 deficiency does not affect band 3 anion transport activity, since uptake of radiolabeled sulfate was similar for control and P4.2-deficient RBCs. Thus, we propose that P4.2 directly participates in strengthening the band 3-cytoskeleton linkage.

Synthesis and characterization of a novel spin-labeled affinity probe of human erythrocyte band 3: characteristics of the stilbenedisulfonate binding site.

A new spin-labeled maleimide derivative of the anion exchange inhibitor 4-4'-diaminodihydrostilbene-2,2'-disulfonate (H2DADS) has been synthesized as a site-specific molecular probe of the stilbenedisulfonate binding site of the anion exchange protein 1 (AE-1; band 3) in human erythrocytes. This probe, SL-H2DADS-maleimide, specifically and covalently labels the Mr 17 kDa integral membrane segment of band 3 with a 1:1 stoichiometry and inhibits essentially 100% of the band 3-mediated anion exchange. The linear V1 EPR spectrum of spin-labeled intact erythrocytes is indicative of a spatially isolated probe which is effectively immobilized on the submicrosecond time scale. Several independent lines of experimental evidence have shown that the nitroxide moiety of SL-H2DADS-maleimide-labeled band 3 is sequestered in a highly protected protein environment. These results are consistent with the observation that the spin-label is rigidly linked to band 3 in a fixed orientation with respect to the membrane normal axis [Hustedt, E. J., & Beth, A. H., (1996) Biochemistry 35, 6944-6954]. The nitroxide moieties of the SL-H2DADS-maleimide-labeled band 3 dimer are greater than 20 A from each other and are also more than 20 A from a monomer-monomer contact surface defined by cross-linking with the spin-labeled reagent BSSDA [bis(sulfo-N-succinimidyl)doxyl-2-spiro-5'-azelate]. These properties make SL-H2DADS-maleimide an extremely useful molecular probe for characterization of the physical properties of the band 3 stilbenedisulfonate binding site, determination of distances between the stilbenedisulfonate site and other segments of band 3, and investigation of the global rotational dynamics of human erythrocyte band 3.

Determination of the orientation of a band 3 affinity spin-label relative to the membrane normal axis of the human erythrocyte.

The orientation of the nitroxide moiety of an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([15N,2H13]-SL-H2DADS-maleimide) covalently coupled at the extracellular stilbenedisulfonate binding site of the human erythrocyte anion exchange protein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (EPR) spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have been obtained for two orthogonal orientations of the sample in the DC magnetic field. Two different methods of analysis have provided very similar values for the angles alpha 1 and beta 1 which uniquely define the orientation of the nitroxide axis frame relative to the membrane normal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the flat cell surface with the remainder, 1-f, isotropically oriented. Simultaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alpha 1 = 58 degrees, and beta 1 = 36 degrees. In the second approach, the EPR spectra of flow-oriented intact erythrocytes labeled with the fatty acid spin-label, [15N,2H12]-5-nitroxyl stearate, have been obtained at the two sample orientations. These two spectra have been used to determine a model-independent distribution of membrane normal orientations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled erythrocytes were then reanalyzed to obtain a second determination of the nitroxide orientation, alpha 1 = 61 degrees and beta 1 = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study is nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST-EPR) studies [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. This result supports the conclusion that the motion observed using ST-EPR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal.

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.

Monitoring DNA dynamics using spin-labels with different independent mobilities.

The electron paramagnetic resonance (EPR) spectra of spin-labeled DNA duplexes, both bound to DEAE-Sephadex and free in solution, have been analyzed. The nitroxide spin-labels are covalently linked to a deoxyuridine residue using either a monoacetylene or diacetylene tether. This difference in tether length produces a dramatic difference in the independent mobility of the nitroxide relative to the DNA. In the case of the monoacetylene tether, the motion of the nitroxide has previously been shown to be tightly coupled to that of the DNA duplex. With the diacetylene tether, there is considerable independent motion of the probe. The diacetylene tether is intended to minimize the possibility of the nitroxide producing a perturbation of the dynamics of DNA. It is demonstrated here that, when coupled via the diacetylene tether, the nitroxide undergoes a rapid uniaxial rotation about the tether. A detailed analysis of the EPR spectrum of duplex DNA in solution, spin-labeled using the diacetylene tether, demonstrates that the motion of the nitroxide can be modeled in terms of this independent uniaxial rotation together with motion of the DNA which is consistent with the global tumbling of the duplex. As was previously found using the monoacetylene tether, there is no evidence of rapid, large-amplitude motions of the base pair in the EPR spectrum of a nitroxide coupled to duplex DNA via the diacetylene tether. This result confirms the small amplitudes of internal motion, local and collective, previously observed in duplex DNA with the monoacetylene-tethered nitroxide.

Protein rotational dynamics investigated with a dual EPR/optical molecular probe. Spin-labeled eosin.

An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in studies of rotational dynamics of the free probe and of the probe when bound noncovalently to bovine serum albumin using the spectroscopic techniques of fluorescence anisotropy decay and electron paramagnetic resonance (EPR) and their long-lifetime counterparts phosphorescence anisotropy decay and saturation transfer EPR. Previous work (Beth, A. H., Cobb, C. E., and J. M. Beechem, 1992. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe. Society of Photo-Optical Instrumentation Engineers. Time-Resolved Laser Spectroscopy III. 504-512) has shown that the spin-label moiety only slightly altered the fluorescence and phosphorescence lifetimes and quantum yields of 5-SLE when compared with 5-SLE whose nitroxide had been reduced with ascorbate and with the diamagnetic homolog 5-acetyleosin. In the present work, we have utilized time-resolved fluorescence anisotropy decay and linear EPR spectroscopies to observe and quantitate the psec motions of 5-SLE in solution and the nsec motions of the 5-SLE-bovine serum albumin complex. Time-resolved phosphorescence anisotropy decay and saturation transfer EPR studies have been carried out to observe and quantitate the microseconds motions of the 5-SLE-albumin complex in glycerol/buffer solutions of varying viscosity. These latter studies have enabled a rigorous comparison of rotational correlation times obtained from these complementary techniques to be made with a single probe. The studies described demonstrate that it is possible to employ a single molecular probe to carry out the full range of fluorescence, phosphorescence, EPR, and saturation transfer EPR studies. It is anticipated that "dual" molecular probes of this general type will significantly enhance capabilities for extracting dynamics and structural information from macromolecules and their functional assemblies.

Measurement of rotational dynamics by the simultaneous nonlinear analysis of optical and EPR data.

In the preceding companion article in this issue, an optical dye and a nitroxide radical were combined in a new dual function probe, 5-SLE. In this report, it is demonstrated that time-resolved optical anisotropy and electron paramagnetic resonance (EPR) data can be combined in a single analysis to measure rotational dynamics. Rigid-limit and rotational diffusion models for simulating nitroxide EPR data have been incorporated into a general non-linear least-squares procedure based on the Marquardt-Levenberg algorithm. Simultaneous fits to simulated time-resolved fluorescence anisotropy and linear EPR data, together with simultaneous fits to experimental time-resolved phosphorescence anisotropy decays and saturation transfer EPR (ST-EPR) spectra of 5-SLE noncovalently bound to bovine serum albumin (BSA) have been performed. These results demonstrate that data from optical and EPR experiments can be combined and globally fit to a single dynamic model.

Motions of short DNA duplexes: an analysis of DNA dynamics using an EPR-active probe.

The dynamics of a series of four DNA duplexes of length 12, 24, 48, and 96 base pairs have been studied using an electron paramagnetic resonance (EPR) active nitroxide spin-label covalently attached to a thymidine located near the center of each duplex. The linear EPR spectra were simulated by solving the stochastic Liouville equation for anisotropic rotational diffusion. The diffusion tensor for global rotation of the duplex was predicted from hydrodynamic theory for a right circular cylinder. All internal motions, assumed to be rapid, are modeled by reduced electron Zeeman and hyperfine tensor anisotropies. Best fit simulations to the data were then obtained by adjusting the total amplitude of all internal dynamics. The local, length-independent and the collective, length-dependent contributions to the internal dynamics were separated by determining the total amplitude of internal motion as a function of duplex length. The major axis of the spin tensors was determined to be tilted 20 degrees from the helix axis. As a result, the spin-label is most sensitive to flexural motions of the DNA duplex. It is found that the global tumbling of duplex is accurately modeled by hydrodynamic theory. The length-independent motion is characterized by a root-mean-squared amplitude of oscillation of 10 degrees in two dimensions at 20 degrees C and has a strong temperature dependence, indicating that the local structure of the DNA changes with temperature. The length dependence of the internal dynamics leads to an estimate of the dynamic flexural persistence length of 2500 +/- 340 A. There was no statistically significant difference between models assuming a harmonic or a square-well local bending potential.