Fast Mapping of Biomolecular Interfaces by Random Spin Labeling (RSL)

Abstract

Random spin labeling (RSL) is a method for rapid mapping of biomolecular interaction surfaces using an interaction partner with SL and an interaction partner enriched in ¹³C or ¹⁵N nuclei for paramagnetic relaxation enhanced NMR-based detection. The SL reaction is conducted in a manner resulting in a heterogeneous reaction product consisting of different populations of the protein carrying a varying number of spin labels at different positions. Preparation of the paramagnetic probe is complete within a few hours and hence much faster than site selective SL. RSL is applicable to tightly interacting systems but shows its particular strength when applied to systems involving weak or transient contacts.     

Operation Everest III: role of plasma volume expansion on VO(2)(max) during prolonged high-altitude exposure.

We hypothesize that plasma volume decrease (DeltaPV) induced by high-altitude (HA) exposure and intense exercise is involved in the limitation of maximal O(2) uptake (VO(2)(max)) at HA. Eight male subjects were decompressed for 31 days in a hypobaric chamber to the barometric equivalent of Mt. Everest (8,848 m). Maximal exercise was performed with and without plasma volume expansion (PVX, 219-292 ml) during exercise, at sea level (SL), at HA (370 mmHg, equivalent to 6, 000 m after 10-12 days) and after return to SL (RSL, 1-3 days). Plasma volume (PV) was determined at rest at SL, HA, and RSL by Evans blue dilution. PV was decreased by 26% (P < 0.01) at HA and was 10% higher at RSL than at SL. Exercise-induced DeltaPV was reduced both by PVX and HA (P < 0.05). Compared with SL, VO(2)(max) was decreased by 58 and 11% at HA and RSL, respectively. VO(2)(max) was enhanced by PVX at HA (+9%, P < 0.05) but not at SL or RSL. The more PV was decreased at HA, the more VO(2)(max) was improved by PVX (P < 0.05). At exhaustion, plasma renin and aldosterone were not modified at HA compared with SL but were higher at RSL, whereas plasma atrial natriuretic factor was lower at HA. The present results suggest that PV contributes to the limitation of VO(2)(max) during acclimatization to HA. RSL-induced PVX, which may be due to increased activity of the renin-aldosterone system, could also influence the recovery of VO(2)(max).     

Properies of tRNAPhe from yeast carrying a spin label on the 3''-terminal. Interaction with yeast phenylalanyl-tRNA Synthetase and elongation factor Tu from Escherichia coli.

The 2-thioketo function of tRNAPhe-C-s2C-A in which the penultimate cytidine residue is replaced by 20thiocytidine can serve as a site of specific attachment of spin label. By alkylation of tRNAPhe-C-s2C-A with iodoacetamide or its spin label derivatives tRNAPhe-C-(acm)s2C-A or tRNAPheC-(SL)s2C-A are formed. The enzymatic phenylalanylation of these tRNAsPhe revealed that the 2-position of the penultimate cytidine can be modified without impairing this enzymatic reaction but there exists a sterical limitation for the subsituent on this position beyond which the tRNAPhe:phenylalanyl-tRNA synthetase recognition is not possible. Both Phe-tRNAPhe-C-(acm)s2C-A as well as Phe-tRNAPhe-C(SL)s2C-A form ternary complexes with EF-Tu.GTP. The part of the 3'-terminus of tRNAPhe where the additional substituents are attached is therefore not involved in the interaction with this elongation factor. This could be also demonstrated by ESR measurements of spin labelled tRNAsPhe. The correlation times, tauc, for tRNAPhe-C-(SL)s2C-A, Phe-tRNAPhe-C-(SL)s2C-A and Phe-tRNAPhe-C-(SL)s2C-A.EF-Tu:GTP are essentially identical indicating that the structure of the 3'-end of tRNAPhe is not influenced significantly by aminoacylation or ternary complex formation.     

饶阳地区三种农田啮齿类头骨形态比较及性二型

关于动物头骨形态方面的研究报道曾受到了较多的关注（Cesani et al., 2003）.头骨形态是动物形态特征的一个重要方面,受遗传和环境因素的共同影响,存在着种间、性别和地理差异（张焱等,1999）.头骨的形态特征不仅是动物生理上的一个基本解剖指标,同时也是动物分类（李晓晨和王廷正,1992）和年龄鉴定（张洁,1989）的重要依据,是动物生态研究的重要指标.对头骨形态的研究,不仅对动物的种间、种内关系（Yom et al.,1999）和生态特征（Barlow et al.,1997）等方面的研究具有重要的参考价值,在动物的地理演化、发育生物学以及进化生态学的研究中也具有十分重要的意义.     

Spin-labelled lutein as a new antioxidant in protection against lipid peroxidation

A new potentially antioxidant compound, spin-labelled lutein (SL-lut), was synthesized and incorporated into egg yolk phosphatidylcholine (EYPC) liposome membrane. The approximate location of nitroxide free radical groups of SL-lut was determined based on electron paramagnetic resonance (EPR) spectra. Then the ability of SL-lut to protect EYPC liposomes against lipid peroxidation (LPO) was compared to the antioxidant effects of lutein and a nitroxide spin label 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (3-CP). Two free radical generation systems were used—a thermal decomposition of 2,2'-azobis (2,4 dimethyl-valeronitrile) (AMVN) and a modified Fenton reaction using ferric-8-hydroxyquinoline (Fe(HQ)₃). Determination of the amount of thiobarbituric acid reactive species (TBARS) was used as a measure of LPO. SL-lut was the most powerful antioxidant, reducing LPO by about 6-times in AMVN-treated liposomes and 7-times in Fe(HQ)₃-treated liposomes. Lutein alone gave only a moderate protection in both systems, while 3-CP was as efficient as SL-lut in the presence of AMVN, but not efficient whatsoever in the presence of Fe(HQ)₃. The results suggest that a nitroxide part of SL-lut plays an important role in enhancing the antioxidant activity of lutein and makes SL-lut a powerful antioxidant efficient under different conditions.     

A spin-label assay for metal ion chelation and complex formation.

The electron spin resonance (ESR) lines of nitroxide spin labels are broadened by electron spin exchange reactions that take place during collisions with paramagnetic ions. The degree of line broadening is greatly reduced when the paramagnetic ion forms a coordination bond with certain functional groups on organic molecules. These observations form the basis for a spin-label assay for metal ion chelation and complex formation. This paper describes the characteristics of such an assay for divalent nickel ions and the spin label TEMPONE (2,2,6,6-tetramethylpiperidone-N-oxyl). The chelation of Ni²⁺ by cysteine and the interaction of Ni²⁺ with sodium dodecyl sulfate micelles and phospholipid vesicles are demonstrated. In addition to monitoring interactions of paramagnetic ions, the assay also allows the detection of interactions of nonparamagnetic ions that compete with the paramagnetic ions for binding sites. A kinetic analysis of competition between Ni²⁺ and Zn²⁺ ions for binding sites on phospholipid vesicles is presented. There are several advantages of the spin-label line-broadening assay compared to other conventional assays for metal chelation and complex formation. The line-broadening assay does not require that the sample be optically clear or chemically defined, it requires only very small quantities of material, it can detect as little as 0.4 to 1 μmol of complexing agent, and it may be utilized in complex biological systems including subcellular organelles and macromolecules.     

Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme–copper site in cytochrome bo 3, E. coli

A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme–copper dinuclear site in Escherichia coli cytochrome bo _3, a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T ₁, of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T ₂, of the dinuclear coupled pair Fe(III)–Cu_B(II) in the oxidized form of QO that is too short to measure directly. In the case of the F′ state, the relaxation properties of the heme–copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu_B(II) coupled by approximately 1.5–3 cm⁻¹ to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine–histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.     

Kinetic analysis-based quantitation of free radical generation in EPR spin trapping

Because short-lived reactive oxygen radicals such as superoxide have been implicated in a variety of disease processes, methods to measure their production quantitatively in biological systems are critical for understanding disease pathophysiology. Electron paramagnetic resonance (EPR) spin trapping is a direct and sensitive technique that has been used to study radical formation in biological systems. Short-lived oxygen free radicals react with the spin trap and produce paramagnetic adducts with much higher stability than that of the free radicals. In many cases, the quantity of the measured adduct is considered to be an adequate measure of the amount of the free radical generated. Although the intensity of the EPR signal reflects the magnitude of free radical generation, the actual quantity of radicals produced may be different due to modulation of the spin adduct kinetics caused by a variety of factors. Because the kinetics of spin trapping in biochemical and cellular systems is a complex process that is altered by the biochemical and cellular environment, it is not always possible to define all of the reactions that occur and the related kinetic parameters of the spin-trapping process. We present a method based on a combination of measured kinetic data for the formation and decay of the spin adduct alone with the parameters that control the kinetics of spin trapping and radical generation. The method is applied to quantitate superoxide trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In principle, this method is broadly applicable to enable spin trapping-based quantitative determination of free radical generation in complex biological systems.     

Assessment of Arsenite,Arsenate, and Chromate Phytotoxicity Based on the Activity of Seed Germination and Growth (Root & Shoot) of Various Plant Seeds

Effects of different concentrations of arsenite, arsenate, and chromate on seed germination, root length (RL), and shoot length (SL) in four seed types, chosen from preliminary tests with eight seed types, were investigated to assess the toxicity of the tested metals. The sensitivities of the four different seed types toward germination, relative RL (RRL), and relative SL (RSL) varied with each metal. In a comparison, the germination of the seeds was more sensitive to the tested metals than the other chosen endpoints (RL and SL). Arsenite was generally more restrictive to all the endpoints (germination, root, and shoot growth) than arsenate and chromate. Lactuca (garden lettuce) was also generally more sensitive to the tested metals than the other seed types. The correlation between RRL and RSL varied depending on the seed type and metal tested. However, significant correlations (r² > 0.85) of these were observed with Lactuca seeds, which appeared to be an optimal plant with respect to the tolerance of the tested metals. The differences in the toxicities of metals toward different plant species should be taken into account in the bioassessment of metals-contaminated sites. Thus, this study encourages the need to combine the three endpoints of various seeds in the evaluation of toxicities of metals.     

Interaction between TOAC free radical and photoexcited triplet chromophores linked to peptide templates

The intramolecular quenching of photoexcited triplet states by free radicals linked to peptide templates was studied by time-resolved electron paramagnetic resonance (EPR) with pulsed laser excitation. The systems investigated are 3(10)-helix forming peptides, having in the amino acid sequence the free radical 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and a triplet precursor, such as Bin, Bpa, or Trp, incorporated at different relative positions. Upon interaction with the excited triplet the TOAC radical spin sublevel populations assume values that differ from the Boltzmann equilibrium values. This spin polarization effect produces EPR lines in emission whose time evolution reflects the triplet quenching rate. In particular, in a series of peptides labeled with Bpa and TOAC at successive positions in the 3(10)-helix, radical-triplet interaction was observed in all cases. However, for the peptide where Bpa and TOAC are at positions 2 and 4 the rate of triplet quenching is lower than for the other peptides in the series. In addition, the radical-excited triplet complex in the quartet spin state was observed in a peptide containing fullerene (C(60)) as a triplet precursor and TOAC.     

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.     

Zero-field splitting of Fe3+ in horseradish peroxidase and of Fe4+ in horseradish peroxidase compound I from electron spin relaxation data.

From the temperature dependence of the Orbach relaxation rate of the paramagnetic center in horseradish peroxidase (HRP), we deduce an excited-state energy of 40.9 +/- 1.1 K. Similar studies on the broad EPR signal of HRP compound I indicate a much weaker Orbach relaxation process involving an excited state at 36.8 +/- 2.5 K. The strength of the Orbach process in HRP-I is weaker than one would normally estimate by 2-4 orders of magnitude. This fact lends support to the model of HRP-I involving a spin 1/2 free radical coupled to a spin 1 Fe4+ heme iron via a weak exchange interaction. Such a system should exhibit an Orbach relaxation process involving delta E, the excited state of the Fe4+ ion, but reduced in strength by (Jyy/delta E)2, where Jyy is related to the strength of the exchange interaction between the two spin systems.     

Use of heme spin-labeling to probe heme environments of alpha and beta chains of hemoglobin.

A spin label attached to a propionic acid group of the heme has been used to probe the heme environment of the alpha and beta chains of hemoglobin in both the subunit and tetrameric forms. The electron paramagnetic resonance (EPR) studies of hemoglobin hybrids in which the spin label is attached to either the alpha- or beta-heme (alpha2SLbeta 2 or alpha2beta2SL) and spin-labeled isolated chains (alphaSL and betaSL) show that: 1) alpha- and beta-hemes have different environments in the tetrameric forms of oxy-, deoxy-, and methemoglobins as well as in isolated single chains; 2) when isolated subunits associate to form hemoglobin tetramers, the environment of the alpha-heme changes more drastically than that of the beta-heme; 3) upon deoxygenation of hemoglobin, the structure in the vicinity of the alpha-heme changes more drastically than that of the beta-heme; and 4) upon the addition of organic phosphates to methemoglobin, the change in the spin state of the heme irons mainly arises from beta-heme. The results demonstrate conclusively that the alpha and the beta subunits of hemoglobin are structurally nonequivalent as are their structural changes as the result of ligation. The relationship of EPR spectrum and structure of hemoglobin is discussed.     

Characterization of a succinyl-CoA radical-cob(II)alamin spin triplet intermediate in the reaction catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.     

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.     

Identification of cis-Acting Nucleotides and a Structural Feature in West Nile Virus 3′-Terminus RNA That Facilitate Viral Minus Strand RNA Synthesis

The 3′-terminal nucleotides (nt) of West Nile virus (WNV) genomic RNA form a penultimate 16-nt small stem-loop (SSL) and an 80-nt terminal stem-loop (SL). These RNA structures are conserved in divergent flavivirus genomes. A previous in vitro study using truncated WNV 3′ RNA structures predicted a putative tertiary interaction between the 5′ side of the 3′-terminal SL and the loop of the SSL. Although substitution or deletion of the 3′ G (nt 87) within the SSL loop, which forms the only G-C pair in the predicted tertiary interaction, in a WNV infectious clone was lethal, a finding consistent with the involvement in a functionally relevant pseudoknot interaction, extensive mutagenesis of nucleotides in the terminal SL did not identify a cis-acting pairing partner for this SSL 3′ G. However, both the sequence and the structural context of two adjacent base pairs flanked by symmetrical internal loops in the 3′-terminal SL were shown to be required for efficient viral RNA replication. Nuclear magnetic resonance analysis confirmed the predicted SSL and SL structures but not the tertiary interaction. The SSL was previously reported to contain one of three eEF1A binding sites, and G87 in the SSL loop was shown to be involved in eEF1A binding. The nucleotides at the bottom part of the 3′-terminal SL switch between 3′ RNA-RNA and 3′-5′ RNA-RNA interactions. The data suggest that interaction of the 3′ SL RNA with eEF1A at three sites and a unique metastable structural feature may participate in regulating structural changes in the 3′-terminal SL.