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

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 (13)C or (15)N 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.     

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.     

Hydroxyl radical production by the iron complex of the hydrolysis product of the antioxidant cardioprotective agent ICRF-187 (dexrazoxane)

SUMMARY

Dexrazoxane (ICRF-187) is now in clinical use for the prevention of doxorubicin-induced cardiotoxicity. This cardiotoxicity is thought to be due to iron-mediated oxidative stress. Dexrazoxane may be acting through its strongly metal ion binding rings-opened hydrolysis product ADR-925 by complexing iron. Since iron-chelates are known to be able to produce hydroxyl radicals, an electron paramagnetic resonance spin trapping study was undertaken to compare the hydroxyl radical-producing ability of the ferrous-ADR-925 complex with that of the ferrous complexes of ethylenediaminetetraacetic acid (EDTA) and the tetraacid analog of ADR-925 (DAPTA). In spectrophotometric studies it was shown that the ferrous-ADR-925 complex underwent aerobic oxidation 87 and 44 times slower than the ferrous complexes of EDTA or 1,2-diaminopropane-N,N,N',N'-tetraacetic acid (DAPTA), respectively. In spite of the much slower oxidation of the ferrous-ADR-925 complex, it was, nonetheless, equally effective in producing hydrogen peroxide-dependent spin adducts. These spin adducts were produced from the reaction of the spin trap with free hydroxyl radical (HO^.), and with a transient iron oxidant with HO^.-like reactivity. Thus, it is concluded that ADR-925 acts by either complexing free iron or iron bound to doxorubicin, and forming a soluble iron complex that is less effective at producing site-specific oxygen radical damage.     

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.     

Studies on electron paramagnetic resonance spectra manifested by a respiratory chain hydrogen carrier.

The high-potential iron-sulfur protein (HiPIP) center of succinate dehydrogenase has an electron paramagnetic resonance (epr) signal in the oxidized form, centered at g = 2.01, and under certain conditions this epr signal is accompanied by absorbances at g = 2.04, g = 1.99, and g = 1.96. These absorbances have been attributed to a spin-spin interaction of paramagnetic species, the semiquinone form of ubiquinone being involved (Ruzicka et al., Proc. Nat. Acad. Sci. USA72, 2886). In the present work this magnetic interaction is studied further; it is concluded that of the three possible species (HiPIP, Flavin H and UQ?H (ubiquinone)) which may interact with UQ?H; a second UQ? most likely partner for the interaction. Nonetheless, the HiPIP center of succinate dehydrogenase also plays a role in the interaction by acting as a “magnetic relaxer” of one or both of the interacting UQ?Hs. The physiological reaction of that part of the ubiquinone pool associated with the succinate dehydrogenase (on the matrix side of the inner mitochondrial membrane) is UQH₂ ? UQ?H + H⁺ + e^?. This is in line with recent postulates of the mechanism of ubiquinone mediation in electron transfer.     

Stacking Interactions between Carbohydrate and Protein Quantified by Combination of Theoretical and Experimental Methods

Carbohydrate – receptor interactions are an integral part of biological events. They play an important role in many cellular processes, such as cell-cell adhesion, cell differentiation and in-cell signaling. Carbohydrates can interact with a receptor by using several types of intermolecular interactions. One of the most important is the interaction of a carbohydrate''s apolar part with aromatic amino acid residues, known as dispersion interaction or CH/π interaction. In the study presented here, we attempted for the first time to quantify how the CH/π interaction contributes to a more general carbohydrate - protein interaction. We used a combined experimental approach, creating single and double point mutants with high level computational methods, and applied both to Ralstonia solanacearum (RSL) lectin complexes with α-l-Me-fucoside. Experimentally measured binding affinities were compared with computed carbohydrate-aromatic amino acid residue interaction energies. Experimental binding affinities for the RSL wild type, phenylalanine and alanine mutants were −8.5, −7.1 and −4.1 kcal.mol⁻¹, respectively. These affinities agree with the computed dispersion interaction energy between carbohydrate and aromatic amino acid residues for RSL wild type and phenylalanine, with values −8.8, −7.9 kcal.mol⁻¹, excluding the alanine mutant where the interaction energy was −0.9 kcal.mol⁻¹. Molecular dynamics simulations show that discrepancy can be caused by creation of a new hydrogen bond between the α-l-Me-fucoside and RSL. Observed results suggest that in this and similar cases the carbohydrate-receptor interaction can be driven mainly by a dispersion interaction.     

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).     

Identification of the divalent metal ion binding domain of myosin regulatory light chains using spin-labelling techniques

By the criterion of their primary structure myosin regulatory light chains belong to the ‘calcium binding protein’ family and are thought to contain domains related to the E-F hand structure found in parvalbumin. However, the presence of deletions and non-conservative substitutions in the regulatory light chains indicates that, of the four domains apparent in their structure, only the first is competent to bind Ca²⁺ or other divalent metal ions. Electron paramagnetic resonance studies were performed in an attempt to provide experimental verification of this hypothesis. The approach is based on the finding that the paramagnetic Mn²⁺ ion substitutes for Ca²⁺ at the divalent metal ion site and that different regulatory light-chain isotypes contain cysteine residues in different domains which may be spin-labelled with a nitroxide derivative. The electron spin interaction between these two paramagnetic centres is a function of the distance of their separation. Clam (Mercenaria mercenaria) regulatory light chain contains a single cysteine residue located near the first domain and, when spin-labelled, the intensity of the nitroxide signal is reduced by 25% on binding one mole of Mn²⁺. Rabbit skeletal regulatory light chain contains two cysteine residues located in the third and fourth domains and no (<5%) interaction is observed when Mn²⁺ binds to spin-labelled derivatives. Qualitatively, these results suggest that domain 1 is the most likely candidate for the Mn²⁺ binding site. A more quantitative evaluation using the Leigh (1970) theory for the dipolar coupling between rigid-lattice electron spins and various models for the regulatory light chain tertiary structure, including that predicted by Kretsinger &; Barry (1975) for the possibly isologous troponin C structure, substantiates this conclusion.     

Hydroxyl and 1-hydroxyethyl free radical detection using spin traps followed by derivatization and gas chromatography—mass spectrometry

Summary

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography—mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl—α—phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, ^.OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of ^.OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

Elucidation of the mechanism of N-demethylation catalyzed by cytochrome P450 monooxygenase is facilitated by exploiting nitrogen-15 heavy isotope effects

¹⁵N heavy isotope effects are especially useful when detail is sought pertaining to the reaction mechanism for the cleavage of a C–N bond. Their potential in assisting to describe the mechanism of N-demethylation of tertiary amines by the action of cytochrome P450 monooxygenase has been investigated. As a working model for the first step, oxidation of the N-methyl group to N-methoxyl, tropine and a cytochrome P450 monooxygenase reaction centre composed of a truncated heme with sulfhydryl as the axial ligand were used. It is apparent that this first step of the reaction proceeds via a hydrogen atom transfer mechanism. Transition states for this step are described for both the high spin (⁴TS_H) and low spin (²TS_H) pathways in both gas and solvation states. Hence, overall normal secondary ¹⁵N KIE could be calculated for the reaction path modeled in the low spin state, and inverse for the reaction modeled in the high spin state. This partial reaction has been identified as the probable rate limiting step. The model for the second step, fission of the C–N bond, consisted of N-methoxylnortropine and two molecules of water. A transition state described for this step, TS_CN, gives a strongly inverse overall theoretical ¹⁵N KIE.     

Autoionization reaction of phosgene (OCC12) studied by electron paramagnetic resonance/spin trapping techniques

The reaction of phosgene with ni-trone spin traps was investigated using electron paramagnetic resonance (EPR)/spin trapping techniques. Evidence for the intermediacy of a carba-moyl monochloride intermediate was obtained. Isotopic substitution of ¹³C-phosgene was employed to verify the hyperfine coupling constant assignments. The implications of these observations on pulmonary damage caused by inhalation of phosgene are mentioned.     

Effects of alkali ions on myosin conformation

We have used two probes to study the effects of alkali ions on the conformation of myosin. One was paramagnetic, the “spin label” N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide, which binds primarily to SH groups; and the other was fluorescent, l-anilino-8-naphthalenesulfonate, which binds to an apolar niche. The bonding of the spin label to myosin was carried out in 0.6M LiCl, 0.6M NaCl, or 0.6M KCl, and the resulting labeled myosin was studied in the same medium in which the myosin was labeled as well as in other alkali chlorides. The electron paramagnetic resonance spectra of the spin label showed that the structure of myosin in the vicinity of the labeled groups differed in the various salts. The protein surface in the region of the labeled groups restricted the rotational freedom of the spin label more in KCl than in any of the other salts. Although ions are known to influence the properties of myosin, our results show that these ions also effect the molecular structure. The fluorescence of l-anilino-8-naphthalenesulfonate, noncovalently attached to myosin in the presence of alkali chlorides, decreased progressively with increasing size of the cations, again showing the protein structure near the probe attachment to be a function of the cation, in the solvent. Ca²⁺ quenched the fluorescence of the bound probe, indicating an interaction between Ca²⁺ and the myosin molecule. The effect of Ca²⁺ on the fluorescence was greatest in KCl.     

Angular dependence of dipole-dipole-Curie-spin cross-correlation effects in high-spin and low-spin paramagnetic myoglobin

The ¹⁵N-HSQC spectra of low-spin cyano-met-myoglobin and high-spin fluoro-met-myoglobin were assigned and dipole-dipole-Curie-spin cross-correlated relaxation rates measured. These cross-correlation rates originating from the dipolar ¹H-¹⁵N interaction and the dipolar interaction between the ¹H and the Curie spin of the paramagnetic center contain long-range angular information about the orientation of the ¹H-¹⁵N bond with respect to the iron-¹H vector, with information measurable up to 11 Å from the metal for the low-spin complex, and between 10 to 25 Å for the high-spin complex. Comparison of the experimental data with predictions from crystal structure data showed that the anisotropy of the magnetic susceptibility tensor in low spin cyano-met-myoglobin significantly influences the cross-correlated dipole-dipole-Curie-spin relaxation rates.     

Revealing binding sites for myeloperoxidase on the surface of human low density lipoproteins

Low density lipoproteins (LDL) of human blood, once oxidized, provoke cholesterol accumulation in cells of arterial wall, which favors the development of atherosclerosis. Oxidative modification of LDL can result from their interaction with hypochlorous acid produced in the halogenation cycle of myeloperoxidase (MPO). On account that MPO is able to form complexes with LDL it seems important to learn the forces promoting such contacts and to spot the likely binding sites for the enzyme on the surface of LDL particles. In this study affinity chromatography on MPO-Sepharose showed that MPO-LDL complexes are uncoupled at ionic strength above 0.3 M NaCl or when pH of solution goes below 3.6. This is an evidence of ionic interaction between MPO and LDL. We used spin probes of lipid nature embedded in phospholipid monolayer so that a variety of distances between the surface of an LDL particle and the paramagnetic center of a spin probes was provided. Since MPO interaction with labeled LDL caused no alteration of EPR spectra it was concluded that lipid components of LDL are not involved in MPO binding. Analysis of Mn²⁺ distribution between LDL surface and the aqueous milieu showed that the surface negative charge of LDL is not considerably changed upon interaction with MPO. It can be suggested that interaction of LDL with MPO does not involve phospholipids that are the principal carriers of the surface charge. Among synthetic oligopeptides with amino acid sequences mimicking those of apoB-100 fragments – ¹EEEMLEN⁷, ⁵³VELEVPQ⁵⁹ and ⁴⁴⁵EQIQDDCTGDED⁴⁵⁶ – only the latter could replace MPO in the complex with LDL. It is concluded that the likely site of interaction with MPO is the amino acid stretch 445–456 of apoB-100 in LDL.     

Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes

Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein–protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes (¹⁵N or ¹³C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model protein–protein complexes, making this approach time consuming and expensive. Here, we show a new strategy that combines a modified pulse sequence (¹H_N-Γ₂-CCLS) with an asymmetric labeling scheme to enable the detection of both intra- and inter-molecular PREs simultaneously using only one sample preparation. We applied this strategy to the non-covalent dimer of ubiquitin. Our method confirmed the previously identified binding interface for the transient di-ubiquitin complex, and at the same time, unveiled the internal structural dynamics rearrangements of ubiquitin upon interaction. In addition to reducing the cost of sample preparation and speed up PRE measurements, by detecting the intra-molecular PRE this new strategy will make it possible to measure and calibrate inter-molecular distances more accurately for both symmetric and asymmetric protein–protein complexes.     

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability^{1, 2}. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation³, and ultimately to oxidative stress leading to cell injury or death⁴. Superoxide radical anion (O₂•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O₂•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O₂•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O₂•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase⁵. Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO⁶. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O₂•- in a process called uncoupling, which is believed to be caused by oxidation of heme⁷ or the co-factor, tetrahydrobiopterin (BH₄)⁸.There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production⁹.The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO¹⁰, the phosphoryl-substituted DEPMPO¹¹, and the ester-substituted, EMPO¹² and BMPO¹³, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O₂•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)₂ is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct¹⁴.     

Potential Regulatory Role of Competitive Encounter Complexes in Paralogous Phosphotransferase Systems

There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS^sugar) and one for nitrogen regulation (PTS^Ntr), that utilize proteins enzyme I^sugar (EI^sugar) and HPr, and enzyme I^Ntr (EI^Ntr) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein–protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EI^Ntr:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI^Ntr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI^Ntr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EI^Ntr. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes.