Magnetization of manganese superoxide dismutase from Thermus thermophilus.

The ground state magnetic properties of manganese superoxide dismutase from Thermus thermophilus in its native and reduced forms have been determined using saturation magnetization data. Parallel EPR measurements were used to verify that commonly encountered paramagnetic impurities were at low concentration relative to the metalloprotein. The native enzyme contains high spin Mn(III) (S = 2) with D = +2.44(5) cm-1 and E/D = 0. The reduced enzyme contains high spin Mn(II) (S = 5/2) with D = +0.50(5) cm-1 and E/D = 0.027. These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds. In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(III) present. The result, epsilon 480 = 950(80) M-1 cm-1 (delta epsilon 480 = 740(60) M-1 cm-1) agrees with the previously reported value of epsilon 480 = 910 M-1 cm-1 found by total manganese determination (Sato, S. and Nakazawa, K. (1978) J. Biochem. 83, 1165-1171). The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed.     

The nickel site in active Desulfovibrio baculatus [NiFeSe] hydrogenase is diamagnetic. Multifield saturation magnetization measurement of the spin state of Ni(II).

The magnetic properties of the nickel(II) site in active Desulfovibrio baculatus (DSM 1743) [NiFeSe] hydrogenase have been measured using the multifield saturation magnetization technique. The periplasmic [NiFeSe] hydrogenase was isolated from bacteria grown in excess selenium in the presence of 57Fe. Saturation magnetization data were collected at three fixed fields (1.375, 2.75, 5.5 tesla) over the temperature range from 2 to 100 K. M?ssbauer and EPR spectroscopies were used to characterize the magnetic state of the two [4Fe-4S] clusters of the enzyme and to quantitate the small amounts of iron impurities present in the sample. The nickel(II) site was found to be diamagnetic (low spin, S = 0). In combination with recent results from extended x-ray absorption fine structure studies, this magnetic state indicates that the nickel(II) site of active D. baculatus [NiFeSe] hydrogenase is five-coordinate.     

Novel NMR tools to study structure and dynamics of biomembranes

Nuclear magnetic resonance (NMR) studies on biomembranes have benefited greatly from introduction of magic angle spinning (MAS) NMR techniques. Improvements in MAS probe technology, combined with the higher magnetic field strength of modern instruments, enables almost liquid-like resolution of lipid resonances. The cross-relaxation rates measured by nuclear Overhauser enhancement spectroscopy (NOESY) provide new insights into conformation and dynamics of lipids with atomic-scale resolution. The data reflect the tremendous motional disorder in the lipid matrix. Transfer of magnetization by spin diffusion along the proton network of lipids is of secondary relevance, even at a long NOESY mixing time of 300 ms. MAS experiments with re-coupling of anisotropic interactions, like the 13C-(1)H dipolar couplings, benefit from the excellent resolution of 13C shifts that enables assignment of the couplings to specific carbon atoms. The traditional 2H NMR experiments on deuterated lipids have higher sensitivity when conducted on oriented samples at higher magnetic field strength. A very large number of NMR parameters from lipid bilayers is now accessible, providing information about conformation and dynamics for every lipid segment. The NMR methods have the sensitivity and resolution to study lipid-protein interaction, lateral lipid organization, and the location of solvents and drugs in the lipid matrix.     

Detection and removal of contaminating hydroxylamines from the spin trap DEPMPO, and re-evaluation of its use to indicate nitrone radical cation formation and S(N)1 reactions

A previous report that the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) allows DEPMPO radical cation formation to be detected via the production of a carbon-centred radical adduct (assigned as the cis-hydroxyethyl species, formed by an intramolecular process) is shown to be incorrect. Rather, this and other paramagnetic species arise from the facile oxidation of trace hydroxylamine impurities present in commercial DEPMPO samples. As a result, techniques for the detection and elimination of such hydroxylamine impurities from DEPMPO solutions were developed and are described; these should prove to be of general use in EPR spin trapping experiments.     

Magnetization hysteresis electron paramagnetic resonance. A new null phase insensitive saturation transfer EPR technique with high sensitivity to slow motion.

In electron paramagnetic resonance (EPR) nonlinear phenomena with respect to magnetic-field modulation are often studied by out-of-phase spectra recordings. The existence of a nonzero out-of-phase signal implies that the EPR signal is phase shifted relative to the modulation signal. This phase shift is called a magnetization hysteresis. The hysteresis angle varies during a sweep through the resonance conditions for a free radical. By recording this variation, a magnetization hysteresis (MH) spectrum results. In practice, a MH spectrum is computer calculated from two EPR spectra detected with a 90 degree difference in phase setting. There is no need for a careful null-phase calibration like that in traditional analysis of nonlinearities. The MH spectra calculated from second harmonic EPR spectra of spin labels were highly dependent on the rotational correlation time. The technique can therefore be used to study slow molecular motion. In the present work MH spectra and Hemminga and deJager's magnitude saturation transfer EPR spectra (Hemminga, M. A., and P. A. deJager, 1981, J. Magn. Reson., 43:324-327) have been analyzed to define parameters that can describe variations in the rotational correlation time. A novel modification of the sample holder and temperature regulation equipment is described.     

Sensitivity optimized HCN and HCNCH experiments for 13C/15N labeled oligonucleotides

Triple resonance HCN and HCNCH experiments used in studies of ¹³C/¹⁵N labeled oligonucleotides include extended evolution periods (typically up to 100 ms) to allow coherence transfer through a complex heteronuclear spin network. Unfortunately, most of the magnetization is lost during the evolution due to fast spin–spin relaxation dominated by one-bond ¹H–¹³C dipolar interaction. As demonstrated recently, the sensitivity of the experiments can be dramatically improved by keeping the spin system in a state of proton–carbon multiple-quantum coherence, which is not affected by the strong dipolar coupling. However, the multiple-quantum coherence is very sensitive to homonuclear as well as long-range heteronuclear interactions. Unwanted magnetization transfer due to these interactions can reduce the sensitivity back to the level of a single-quantum experiment and, for some spin moieties, even eliminate the signal completely. In the present paper we show that a modified HCN scheme that refocuses the interfering coherences improves sensitivity routinely by a factor of 1.5 to 4 over a nonselective experiment. In addition, novel multiple-quantum 2D and 3D HCNCH experiments with substantially enhanced sensitivity are presented.     

Improved distance analysis in RNA using network-editing techniques for overcoming errors due to spin diffusion

Multispin magnetization transfer, or spin diffusion, is a significant source of error in NOESY-derived distance measurements for the determination of nucleic acid solution structures. The BD-NOESY and CBD-NOESY experiments, which allow the measurement of interproton distances with greatly reduced contributions from spin diffusion, have been adapted to structural analysis in RNA oligonucleotides. The techniques are applied to a lead-dependent ribozyme (LZ2). We demonstrate the measurement of both aromatic proton–aromatic proton NOEs free of spin diffusion involving the intervening ribose moieties and aromatic proton–ribose proton NOEs free of the efficient cross-relaxation within the ribose ring. In LZ2, the accuracy and precision of the resulting distances are significantly improved. We also find that, by allowing the use of longer mixing times with greater sensitivity, the experimental attenuation of spin diffusion in RNA increases the distance range of interactions that can be analyzed. This effect permits measurement of important long-range distances in LZ2 that are not accessible with standard techniques. Thus, these techniques allow the simultaneous optimization of the number, accuracy, and precision of distance constraints used for RNA structure determinations.     

Synchrotron radiation for direct analysis of metalloproteins on electrophoresis gels

Metalloproteomics requires analytical techniques able to assess and quantify the inorganic species in metalloproteins. The most widely used methods are hyphenated techniques, based on the coupling of a high resolution chromatographic method with a high sensitivity method for metal analysis in solution. An alternative approach is the use of methods for solid sample analysis, combining metalloprotein separation by gel electrophoresis and direct analysis of the gels. Direct methods are based on beam analysis, such as lasers, ion beams or synchrotron radiation beams. The aim of this review article is to present the main features of synchrotron radiation based methods and their applications for metalloprotein analysis directly on electrophoresis gels. Synchrotron radiation X-ray fluorescence has been successfully employed for sensitive metal identification, and X-ray absorption spectroscopy for metal local structure speciation in proteins. Synchrotron based methods will be compared to ion beam and mass spectrometry for direct analysis of metalloproteins in electrophoresis gels.     

Proton NMR spin grouping and exchange in dentin.

The nuclear magnetic resonance spin-grouping technique has been applied to dentin from human donors of different ages. The apparent T2, T1, and T1 rho have been determined for natural dentin, for dentin which has been dried in vacuum, and for dried dentin which has been rehydrated in an atmosphere with 75% relative humidity. All apparent spin relaxation has been analyzed for exchange between the spin groups in which the dentin protons exist; the analyses incorporate the results of selective inversion recovery T1 measurements which better probe the effects of exchange. The exchange analyses of the high fields and rotating frame spin-lattice relaxation have also been correlated to determine uniquely the inherent relaxation parameters of the proton spin groups constituting the dentin magnetization. The natural dentin contains protons on water, protein, and hydroxy apatite; these spins contribute 50%, 45%, and 5% to the total dentin proton magnetization, respectively. The water exists in three distinct environments, the dynamics of each environment has been modeled. In the natural dentin 30% of the water undergoes uni-axial reorientation. 52% of the water has similar relaxation characteristics to bound water hydrating a large molecule, and the majority of the remaining water acts as bulk water undergoing isotropic reorientation. The results are independent of the age of the donor.     

Inelastic neutron scattering investigations of molecular nanomagnets

Inelastic neutron scattering, probing the temporal spin–spin correlation at the microscopic scale, is a powerful technique to study the magnetic behaviour of molecular nanomagnets. Experiments at different energy scales and different energy-transfer resolution allow precise determinations of the parameters defining the effective Hamiltonians used to model the diverse physical properties exhibited by this class of materials. The intrinsic disadvantage of the technique (low flux, requiring a sample mass in the gram scale) is over-compensated by the large amount of information that can be straightforwardly extracted. Zero-field splittings and exchange interactions can be determined with a large degree of confidence, shedding light on important issues such as magnetization tunnelling in giant-spin clusters, or the occurrence of quantum coherence phenomena and their consequences on macroscopic observables.     

Magnetization and electron paramagnetic resonance studies of reduced uteroferrin and its "EPR-silent" phosphate complex

The exchange coupling of reduced uteroferrin has been measured (19.8(5) cm-1 S1.S2) using recently developed techniques for studying metalloprotein magnetization. A spin Hamiltonian describing the coupled binuclear Fe(II).Fe(III) center has been used to fit the low and high field magnetization data, the EPR g values, and the highly anisotropic effective hyperfine tensor of the ferric site. The exchange coupling of the phosphate complex of reduced uteroferrin has also been measured (6.0(5) cm-1 S1.S2) using the same techniques. The smaller exchange coupling of the phosphate complex is comparable with the zero field splittings of the iron sites. This results in increased sensitivity of the system g values (found by calculation from the spin Hamiltonian) to variations of the zero field splitting parameters arising from heterogeneities in the protein microenvironment. Consequently, there is a very significant (9-fold) increase in the "effective g strain" of the system compared to the situation in the absence of phosphate. This, together with the larger g anisotropy (g = (1.06, 1.51, 2.27)), gives rise to an EPR signal for the phosphate complex of reduced uteroferrin which is extremely broad and difficult to detect but which has now been identified for the first time.     

What Is the Primary Cause of Individual Differences in Contrast Sensitivity?

One of the primary objectives of early visual processing is the detection of luminance variations, often termed image contrast. Normal observers can differ in this ability by at least a factor of 4, yet this variation is typically overlooked, and has never been convincingly explained. This study uses two techniques to investigate the main source of individual variations in contrast sensitivity. First, a noise masking experiment assessed whether differences were due to the observer’s internal noise, or the efficiency with which they extracted information from the stimulus. Second, contrast discrimination functions from 18 previous studies were compared (pairwise, within studies) using a computational model to determine whether differences were due to internal noise or the low level gain properties of contrast transduction. Taken together, the evidence points to differences in contrast gain as being responsible for the majority of individual variation across the normal population. This result is compared with related findings in attention and amblyopia.     

Structural Analysis of Metal Sites in Proteins: Non-heme Iron Sites as a Case Study

In metalloproteins, the protein environment modulates metal properties to achieve the required goal, which can be protein stabilization or function. The analysis of metal sites at the atomic level of detail provided by protein structures can thus be of benefit in functional and evolutionary studies of proteins. In this work, we propose a structural bioinformatics approach to the study of metalloproteins based on structural templates of metal sites that include the PDB coordinates of protein residues forming the first and the second coordination sphere of the metal. We have applied this approach to non-heme iron sites, which have been analyzed at various levels. Templates of sites located in different protein domains have been compared, showing that similar sites can be found in unrelated proteins as the result of convergent evolution. Templates of sites located in proteins of a large superfamily have been compared, showing possible mechanisms of divergent evolution of proteins to achieve different functions. Furthermore, template comparisons have been used to predict the function of uncharacterized proteins, showing that similarity searches focused on metal sites can be advantageously combined with typical whole-domain comparisons. Structural templates of metal sites, finally, may constitute the basis for a systematic classification of metalloproteins in databases.     

Synthesis of chromophoric, spin label enzyme substrates useful for cryoenzymology.

The spin label nitroxide derivative 3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oic acid has been synthesized and characterized by chemical methods. It is a useful intermediate in the preparation of a new class of chromophoric spin label substrates for enzyme studies, as shown by the synthesis of O-3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oyl-L-beta-phenyllactic acid, a specific ester substrate of bovine pancreatic carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2). Kinetic parameters of the esterolytic reaction are conveniently determined by UV spectrophotometric methods, and a reaction intermediate can be stabilized in fluid cryosolvent mixtures at subzero temperatures. Results are presented of preliminary electron spin resonance studies to demonstrate that structural relationships of the spin label substrate in a catalytically active configuration to active site residues can be determined for this low temperature-stabilized reaction intermediate. This substrate thus demonstrates the utility of this new class of spin label derivatives for characterization of enzyme reaction intermediates stabilized by cryoenzymologic techniques.     

Electron paramagnetic resonance and saturation transfer electron paramagnetic resonance studies on erythrocytes from goats with and without heritable myotonia

Summary Erythrocytes from myotonic goats, an animal model of heritable myotonia, and normal goats were studied using electron paramagnetic resonance (EPR) and saturation transfer electron paramagnetic resonance (ST-EPR) spin labeling techniques. Three fatty acid spin labels with the nitroxide moiety at progressively greater distances from the carboxyl group were used to monitor different regions within the erythrocyte membrane. Since spin labels have been shown to induce hemolytic and morphologic alterations in erythrocytes, conditions for minimizing these alterations were first defined by hemolysis studies and scanning electron microscopy. Using these defined conditions for our studies we observed no significant differences in any of the EPR or ST-EPR parameters for normal and myotomic goat erythrocytes with any of the fatty acid spin labels used. Our results do not support the theory that myotonia is the result of a generalized membrane defect characterized by increased membrane fluidity as determined by fatty acid spin labels.     

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.     

Violet laser diodes in flow cytometry: an update.

INTRODUCTION: In previous studies we and others have demonstrated the usefulness of violet laser diodes (VLDs) as replacement laser sources for krypton-ion lasers on stream-in-air cytometers. Previously available VLDs had a maximum available power of less than 25 mW; this was sufficient for excitation of densely labeled cell surface antigens using fluorochromes such as Cascade Blue or Pacific Blue, but may have been insufficient for applications requiring higher levels of photon saturation, such as low-level expression of Cyan Fluorescent Protein (ECFP) in CFP-YFP FRET applications. In this follow-up study, we have tested more powerful VLDs emitting at 55 mW, and a beam-merged dual module VLD with 100 mW combined output, for their ability to excite a variety of violet-excited fluorochromes, including CFP. METHODS: A dual module VLD (two linear polarized VLDs with their beams merged by a polarized beam combiner) emitting at 404 nm was mounted on a BD FACSVantage DiVa stream-in-air cytometer. The individual polarized 55 mW beams or the 100 mW combined beams were used to analyze PBMCs labeled with the violet-excited probes Cascade Blue, Alexa Fluor 405, Cascade Yellow and Pacific Orange dyes. Violet-excited fluorescent microsphere mixtures with decreasing fluorescence levels were also used to detect the minimum sensitivity threshold and precision of these lasers. VLD excitation on a gel-coupled cuvette flow cytometer was used as a sensitivity baseline. RESULTS: The dual module 100 mW VLD gave both sensitivity and precision levels approaching that observed for lower-power sources on a cuvette cytometer. Single polarized VLD modules at 55 mW gave slightly decreased sensitivity for the microspheres standards and all the tested fluorochromes compared to the 100 mW source. CONCLUSIONS: While 55 mW laser sources performed adequately in the stream-in-air format, increasing the power to 100 mW did give a small but detectable increase in instrument sensitivity. This sensitivity level approached that of cuvette systems.     

A spectroelectrochemical study of microperoxidase at bare and gold-plated RVC thin-layer electrodes

Direct electrochemistry of microperoxidase (the heme-undecapeptide from cytochrome c) has been followed at a bare and a gold plated RVC thin-layer electrode, using the spectropotentiostatic method or voltabsorptometry. Both techniques yield 'clean' and undistorted signals; their analysis easily provides quantitative information for the electrochemical parameters of microperoxidase and shows that spectroelectrochemistry is a powerful method to study the redox behavior of metalloproteins or their active site fragments.