A spin-labeled abasic DNA substrate for AP endonuclease.

We report the first observation of a spin-labeled ds 23-mer oligonucleotide by high-field electron spin resonance (ESR) and demonstrate that it interacts with AP endonuclease, the key enzyme in DNA abasic site repair. The spin labeled 23-mer with a U at position 12 of the upper strand is processed by uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, AP endo binds and cleaves when the label is 3' but not 5' to the abasic site. These results confirm that the disposition of the bases immediately upstream of the abasic site is particularly critical for cleavage by AP endo, and establish that DNA-protein interactions in this important enzyme can be examined using spin-labeled substrates.

Jones matrix formalism for quasioptical EPR.

The Jones matrix formalism that has been used to analyze quasioptical millimeter-wave circuits is extended for specific application to high-frequency electron paramagnetic resonance (EPR). A survey of general expressions for Jones matrices of elements commonly used in quasioptical EPR spectrometers is given. The Jones matrix representations of quasioptical transmission and reflection cavities are derived, and their relationship to the equivalent circuit and transmission line representations used for conventional EPR cavities is demonstrated. The formalism is applied to selected quasioptical EPR spectrometer designs and experimental tests of the formalism are presented for two configurations of a quasioptical spectrometer operating at 220 GHz.

Dynamics and ordering in a spin-labeled oligonucleotide observed by 220 GHz electron paramagnetic resonance.

The dynamics of a newly synthesized cytosine spin-label and the spin-labeled pentamer TTC*TT have been observed by high-frequency (220 GHz) electron paramagnetic resonance (EPR) in aqueous solution at ambient temperature using only nanomolar amounts of spin-label. Temperature studies were carried out for both labeled species in buffer containing glycerol. The motion of the spin-labeled monomer could be fitted using a model of fully anisotropic rotation (FAR) over the entire temperature range studied. In the single-stranded pentamer, the high-field spectra are best interpreted using a model of microscopic ordering with macroscopic disorder (MOMD) with the probe in a highly nonpolar environment. The observed local order parameters of 0.60-0.70 suggest a micelle-like structure in which the label is tightly packed with the hydrophobic bases. These preliminary studies illustrate how the excellent orientation selectivity of high-field EPR provides new dynamic information about local base motions in DNA, and also how high-field EPR of spin-labels allows one to discriminate accurately between the effects of local versus global motions in spin-labeled macromolecules.

ESR studies of spin-labeled membranes aligned by isopotential spin-dry ultracentrifugation: lipid-protein interactions.

Electron spin resonance (ESR) studies have been performed on spin-labeled model membranes aligned using the isopotential spin-dry ultracentrifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopotential surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainable using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoylphosphatidylcholine (DPPC) model membranes compares favorably with that obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of macroscopically aligned membrane samples that may be investigated by ESR. We report the first ESR study of an integral membrane protein, bacteriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol% BR/DPPC membranes with CSL probe show two distinct components, primarily in the gel phase, which can be attributed to bulk and boundary regions of the bilayer. The boundary regions show sharply decreased molecular ordering and spectral effects comparable to those observed from 2 mol% GA/DPPC membranes. The boundary regions for both BR and GA also exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the disordering effect as evidenced by the reduced populations of the disordered components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptide- and protein-containing membranes reveals a new component with increased ordering of the lipids associated with the peptide or protein. This increase coincides with a broad endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the lipid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have helped to clarify the spectral interpretations. It is found that the simulations of ESR spectra from CSL in the gel phase for all the lipid membranes studied could be significantly improved by utilizing a model with CSL molecules existing as both hydrogen-bonded to the bilayer interface and non-hydrogen-bonded within the bilayer.

An electron spin resonance study of interactions between phosphatidylcholine and phosphatidylserine in oriented membranes.

A detailed electron spin resonance (ESR) study of mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and phosphatidylserine (POPS) in oriented multilayers in the liquid crystalline phase is reported with the purpose of characterizing the effects of headgroup mixing on the structural and dynamical properties of the acyl chains. These studies were performed over a range of blends of POPC and POPS and temperatures, utilizing the spin-labeled lipids 16-phosphatidylcholine and 5-phosphatidylcholine as well as cholestane (CSL). The ESR spectra were analyzed by nonlinear least-squares fitting using detailed spectral simulations. Whereas CSL shows almost no variation in ordering and rotational dynamics versus mole fraction POPS, (i.e. XPS), and 5-PC shows small effects, the weakly ordered end-chain labeled 16-PC shows large relative effects, such that the orientational order parameter, S is at a minimum for XPS = 0.5 where it is about one-third the value observed for XPS = 0 and 1. This is directly reflected in the ESR spectrum as a substantial variation in the hyperfine splitting with XPS. The least-squares analysis also shows a reduction in rotational diffusion coefficient, R perpendicular by a fractor of 2 for XPS = 0.5 and permits the estimation of S2, the ordering parameter representing deviations from cylindrically symmetric alignment. These results are contrasted with 2H NMR studies which were insensitive to effects of mixing headgroups on the acyl chains. The ESR results are consistent with a somewhat increased disorder in the end-chain region as well as a small amount of chain tilting upon mixing POPC and POPS. They demonstrate the high sensitivity of ESR to subtle effects in chain ordering and dynamics.

250-GHz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes.

Rigid-limit 250-GHz electron spin resonance (FIR-ESR) spectra have been studied for a series of phosphatidylcholine spin labels (n-PC, where n = 5, 7, 10, 12, 16) in pure lipid dispersions of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as well as dispersions of DPPC containing the peptide gramicidin A (GA) in a 1:1 molar ratio. The enhanced g-tensor resolution of 250-GHz ESR for these spin labels permitted a careful study of the nitroxide g-tensor as a function of spin probe location and membrane composition. In particular, as the spin label is displaced from the polar head group, Azz decreases and gxx increases as they assume values typical of a nonpolar environment, appropriate for the hydrophobic alkyl chains in the case of pure lipid dispersions. The field shifts of spectral features due to changes in gxx are an order of magnitude larger than those from changes in Azz. The magnetic tensor parameters measured in the presence of GA were characteristic of a polar environment and showed only a very weak dependence of Azz and gxx on label position. These results demonstrate the significant influence of GA on the local polarity along the lipid molecule, and may reflect increased penetration of water into the alkyl chain region of the lipid in the presence of GA. The spectra from the pure lipid dispersions also exhibit a broad background signal that is most significant for 7-, 10-, and 12-PC, and is more pronounced in DPPC than in POPC. It is attributed to spin probe aggregation yielding spin exchange narrowing. The addition of GA to DPPC essentially suppressed the broad background signal observed in pure DPPC dispersions.

Thermodynamics and dynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state: effects of water.

A method for obtaining the thermodynamic activity of each membrane component in phosphatidylcholine (PC)/cholesterol mixtures, that is based upon ESR spin labeling is examined. The thermodynamic activity coefficients, gamma PC and gamma chol, for the PC and cholesterol, respectively, are obtained from the measured orientational order parameters, SPC and S(chol), as a function of cholesterol content for a spin-labeled PC and the sterol-type cholestane spin probe (CSL), respectively, and the effects of water concentration are also considered. At water content of 24 weight%, the thermodynamics of DMPC/cholesterol/water mixtures in the liquid-crystalline state may be treated as a two-component solution ignoring the water, but at lower water content the role of water is important, especially at lower cholesterol concentrations. At lower water content (17 wt%), gamma chol decreases with increasing cholesterol content which implies aggregation. However, at higher water content (24 wt%), gamma chol is found initially to increase as a function of cholesterol content before decreasing at higher cholesterol content. This implies a favorable accommodation for the cholesterol in the membrane at high water and low cholesterol content. Good thermodynamic consistency according to the Gibbs-Duhem equation was obtained for gamma PC and gamma chol at 24 wt% water. The availability of gamma chol (and gamma PC) as a function of cholesterol concentration permits the estimate of the boundary for phase separation. The rotational diffusion coefficients of the labeled PC and of CSL were also obtained from the ESR spectra. A previously proposed universal relation for the perpendicular component of the rotational diffusion tensor, R perpendicular, for CSL in PC/cholesterol mixtures (i.e., R perpendicular = R0 perpendicular exp(-AS2chol/RT)) is confirmed. A change in composition of cholesterol or of water for DMPC/cholesterol/water mixtures affects R perpendicular only through the dependence of S(chol) on the composition. In particular, the amount of water affects the membrane fluidity, monitored by R perpendicular for CSL, solely by the structural changes it induces in the membrane for the compositions studied. Rotational diffusion for the labeled PC is found to be more complex, most likely due to the combined action of the internal modes of motion of the flexible chain and of the overall molecular reorientation.

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.

Correlation of paramagnetic states and molecular structure in bacterial photosynthetic reaction centers: the symmetry of the primary electron donor in Rhodopseudomonas viridis and Rhodobacter sphaeroides R-26.

The orientation of the principal axes of the primary electron donor triplet state measured in single crystals of photosynthetic reaction centers is compared to the x-ray structures of the bacteria Rhodobacter (Rb.) sphaeroides R-26 and Rhodopseudomonas (Rps.) viridis. The primary donor of Rps. viridis is significantly different from that of Rb. sphaeroides. The measured directions of the axes indicate that triplet excitation is almost completely localized on the L-subunit half of the dimer in Rps. viridis but is more symmetrically distributed (approximately 63% on the L half of the special pair and approximately 37% on the M half) on the dimeric donor in Rb. sphaeroides R-26. The large reduction of the zero field splitting parameters relative to monomeric bacteriochlorophyll triplet in vitro suggests significant participation of asymmetrical charge transfer electronic configurations in the special pair triplet state of both organisms (approximately 23% in Rps. viridis and approximately 13% in Rb. sphaeroides).

Magnetic characterization of the primary state of bacterial photosynthesis.

The results of reaction yield-detected magnetic resonance (RYDMR) experiments carried out on modified bacterial photosynthetic reaction centers (RCs) are interpreted in terms of a model that assigns the initial charge-separated radical ion-pair state, P(F), as the carrier of the spectrum. The radical pair theory, which has been invoked to explain magnetic field effects in RCs, was significantly expanded to take into consideration the electron dipole-dipole interaction. It is shown that this is the largest interaction between the components of the radical ion pair. Quantum statistical calculations are described simulating the RYDMR spectra and low-field effects in quinone-depleted RCs. The experimental data on which the simulations are based are (i) the magnitude of the field effect at 3,000 G, (ii) the field at which 0.5 of the maximal field effect is observed, (iii) the P(F) population as a function of time at zero magnetic field, (iv) the RYDMR linewidth for low microwave field strength, (v) the RYDMR intensity and width as a function of microwave field, and (vi) the maximum RYDMR intensity at H(I) approximately 2J. With this information it was found possible to characterize P(F) in terms of four parameters, two containing structural information and two with kinetic implications. These are the dipole-dipole interaction, D = -47 +/- 10 x 10(-4) cm(-1); the exchange interaction, J = -7.5 +/- 1.9 x 10(-4) cm(-1); and the inverse rate constants of the decay of the radical pair states with singlet and triplet spin functions, respectively, k(S) (-1) = 15 +/- 4 nsec and k(T) (-1) = 1.8 +/- 0.2 nsec. The structural and dynamic implications of these parameters are discussed.

Magnetic resonance spectroscopy of the primary state, P, of bacterial photosynthesis.

We have obtained the magnetic resonance spectrum of the radical pair state P(F) by using reaction yield detected magnetic resonance spectroscopy. The magnetic resonance spectrum is quite sensitive to the local environment of P(F). The data place limits on the lifetime of triplet P(F) and the distance of charge separation.