Mapping electron paramagnetic resonance spin label conformations by the simulated scaling method

In order to efficiently simulate spin label behavior when attached to the protein backbone we developed a novel approach that enhances local conformational sampling. The simulated scaling (SS) approach (Li, H., et al. J. Chem. Phys. 2007, 126, 24106) couples the random walk of a potential scaling parameter and molecular dynamics in the framework of hybrid Monte Carlo. This approach allows efficient barrier crossings between conformations. The method retains the thermodynamic detailed balance allowing for determination of relative free energies between various conformations. The accuracy of our method was validated by comparison with the recently resolved X-ray crystal structure of a spin labeled T4 lysozyme in which the spin label was in the interior of the protein. Consistent potentials of mean force (PMF) are obtained for the spin label torsion angles to illustrate their behavior in various protein environments: surface, semiburied, and buried. These PMFs reflect the experimentally observed trends and provide the rationale for the spin label dynamics. We have used this method to compare an implicit and explicit solvent model in spin label modeling. The implicit model, which is computationally faster, was found to be in excellent agreement with the explicit solvent treatment. Based on this collection of results, we believe that the presented approach has great potential in the general strategy of describing the behavior of the spin label using molecular modeling and using this information in the interpretation of EPR measurements in terms of protein conformation and dynamics.     

Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution

Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized.     

Spectroscopic selection of distance measurements in a protein dimer with mixed nitroxide and Gd3+ spin labels

The pulse DEER (Double Electron-Electron Resonance) technique is frequently applied for measuring nanometer distances between specific sites in biological macromolecules. In this work we extend the applicability of this method to high field distance measurements in a protein assembly with mixed spin labels, i.e. a nitroxide spin label and a Gd(3+) tag. We demonstrate the possibility of spectroscopic selection of distance distributions between two nitroxide spin labels, a nitroxide spin label and a Gd(3+) ion, and two Gd(3+) ions. Gd(3+)-nitroxide DEER measurements possess high potential for W-band long range distance measurements (6 nm) by combining high sensitivity with ease of data analysis, subject to some instrumental improvements.     

Ox-SLIM: Synthesis of and Site-Specific Labelling with a Highly Hydrophilic Trityl Spin Label

The combination of pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) with site-directed spin labelling is a powerful tool in structural biology. Rational design of trityl-based spin labels has enabled studying biomolecular structures at room temperature and within cells. However, most current trityl spin labels suffer either from aggregation with proteins due to their hydrophobicity, or from bioconjugation groups not suitable for in-cell measurements. Therefore, we introduce here the highly hydrophilic trityl spin label Ox-SLIM. Engineered as a short-linked maleimide, it combines the most recent developments in one single molecule, as it does not aggregate with proteins, exhibits high resistance under in-cell conditions, provides a short linker, and allows for selective and efficient spin labelling via cysteines. Beyond establishing synthetic access to Ox-SLIM, its suitability as a spin label is illustrated and ultimately, highly sensitive PDS measurements are presented down to protein concentrations as low as 45 nm resolving interspin distances of up to 5.5 nm.     

Conformationally Restricted Isoindoline‐Derived Spin Labels in Duplex DNA: Distances and Rotational Flexibility by Pulsed Electron–Electron Double Resonance Spectroscopy

Three structurally related isoindoline‐derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation‐dependent pulsed electron–electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ^ExIm U , was synthesized and incorporated into DNA oligonucleotides. ^ExIm U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N?O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline‐based spin label to a uridine base. Continuous‐wave (CW) EPR measurements of ^ExIm U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ^Im U , which has restricted mobility due to an intramolecular hydrogen bond. The X‐band CW‐EPR spectra of ^ExIm U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ^Im U , ^Ox U , and ^ExIm U in duplex DNA showed a strong angular dependence for ^Im U , a medium dependence for ^Ox U , and no orientation effect for ^ExIm U . Thus, precise distances can be extracted from ^ExIm U without having to take orientational effects into account.     

Selective High‐Resolution Detection of Membrane Protein–Ligand Interaction in Native Membranes Using Trityl–Nitroxide PELDOR

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero‐oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro–electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter‐subunit interaction and the intra‐subunit conformational changes in hetero‐oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein–ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.     

Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side-Directed Spin Labeling of Proteins

Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self-aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time T_M for HSA labeled with OX063 is 6.3 μs at 50 K, the longest yet obtained with a TAM-based spin label. The lowered lipophilicity also decreases side products in the labeling reaction.     

Light‐Induced Porphyrin‐Based Spectroscopic Ruler for Nanometer Distance Measurements

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin‐labeling method based on the photoexcited triplet state (S=1). Four‐pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N‐terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8–8 nm. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep envelope modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels. The upper limit is evaluated and found to be around 8 nm. The PELDOR‐derived distances are in excellent agreement with theoretical predictions. We demonstrate that high sensitivity is acquired using the triplet state as a spin label by comparison with Cu(II)–porphyrin analogues. The new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the properties of the porphyrin triplet state.     

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid,Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long‐range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein‐backbone structure. Herein we present the double‐histidine (dHis) Cu²⁺‐binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X‐ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu²⁺ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein‐backbone structure and flexibility.     

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid,Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long‐range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein‐backbone structure. Herein we present the double‐histidine (dHis) Cu²⁺‐binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X‐ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu²⁺ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein‐backbone structure and flexibility.     

Measurement of large distances in biomolecules using double-quantum filtered refocused electron spin-echoes

It is shown how the new technique of double-quantum filtered refocused electron spin-echoes is a significant improvement over double-quantum coherence ESR, since it increases the experimental acquisition time. This enables the measurement of longer distances in bilabeled biomolecules. The method is demonstrated on a long double-stranded A-type RNA, spin labeled at both ends. The measured distance of 72 A is in excellent agreement with molecular modeling.     

Measurement of Angstrom to Nanometer Molecular Distances with 19F Nuclear Spins by EPR/ENDOR Spectroscopy

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using ¹⁹F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the ¹⁹F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.     

Stochastic modeling of CW-ESR spectroscopy of [60]fulleropyrrolidine bisadducts with nitroxide probes

In this work, we address the interpretation of continuous wave electron spin resonance (CW-ESR) spectra of fulleropyrrolidine bisadducts with nitroxide addends. Our approach is based on a definition of the spin Hamiltonian which includes exchange and dipolar interactions and on a complete numerical solution of the resulting stochastic Liouville equation, with inclusion of diffusive rotational dynamics. CW-ESR spectra are simulated for a series of C60 bisadducts made up of four trans isomers and the equatorial isomer. A nonlinear least-squares fitting procedure allows extraction directly from the available experimental spectra of a wide range of parameters, namely interprobe relative distances, diffusion tensors, and values of the exchange parameter J. Results are in good agreement with previous, more phenomenological estimates, proving that the combination of sensitive ESR spectroscopy based on multiple spin labeling with nitroxide radicals and sophisticated modeling can be highly helpful in providing structural and dynamic information on molecular systems.     

Conformational flexibility of DNA

Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.     

Using Markov models to simulate electron spin resonance spectra from molecular dynamics trajectories

Simulating electron spin resonance (ESR) spectra directly from molecular dynamics simulations of a spin-labeled protein necessitates a large number (hundreds or thousands) of relatively long (hundreds of nanoseconds) trajectories. To meet this challenge, we explore the possibility of constructing accurate stochastic models of the spin label dynamics from atomistic trajectories. A systematic, two-step procedure, based on the probabilistic framework of hidden Markov models, is developed to build a discrete-time Markov chain process that faithfully captures the internal spin label dynamics on time scales longer than about 150 ps. The constructed Markov model is used both to gain insight into the long-lived conformations of the spin label and to generate the stochastic trajectories required for the simulation of ESR spectra. The methodology is illustrated with an application to the case of a spin-labeled poly alanine alpha helix in explicit solvent.     

High-field EPR and ESEEM investigation of the nitrogen quadrupole interaction of nitroxide spin labels in disordered solids: toward differentiation between polarity and proticity matrix effects on protein function

The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.     

Simulating electron spin resonance spectra of nitroxide spin labels from molecular dynamics and stochastic trajectories

Simulating electron spin resonance spectra of nitroxide spin labels from motional models is necessary for the quantitative analysis of experimental spectra. We present a framework for modeling the spin label dynamics by using trajectories such as those from molecular dynamics (MD) simulations combined with stochastic treatment of the global protein tumbling. This is achieved in the time domain after two efficient numerical integrators are developed: One for the quantal dynamics of the spins and the other for the classical rotational diffusion. For the quantal dynamics, we propagate the relevant part of the spin density matrix in Hilbert space. For the diffusional tumbling, we work with quaternions, which enables the treatment of anisotropic diffusion in a potential expanded as a sum of spherical harmonics. Time-averaging arguments are invoked to bridge the gap between the smaller time step of the MD trajectories and the larger time steps appropriate for the rotational diffusion and/or quantal spin dynamics.     

Aggregation modes of the spin mono-labeled tylopeptin B and heptaibin peptaibiotics in frozen solutions of weak polarity as studied by PELDOR spectroscopy

The X band PELDOR spectroscopy was used to investigate the magnetic dipole-dipole interactions in glassy solutions of nitroxide mono-labeled tylopeptin B and heptaibin peptaibiotics at 77 K. Specifically, a study was performed of the tylopeptin B peptides labeled at either position 3, 8, or 13, denoted as T3, T8, and T13, respectively. The heptaibin analogs labeled at either position 2 or 14, denoted as H2 and H14, respectively, were also investigated. It was shown that in frozen glassy peptide solutions in methanol, the spin labels are randomly distributed over the solvent volume. This result points to the absence of specific dipolar interactions between the peptides under these conditions. However, peptide aggregation was detected in weakly polar methanol/toluene environments. To study the properties of the resulting aggregates, we examined the depth of modulation for the PELDOR traces as a function of the concentration of the peptides in solution and the distances between the spin labels in the aggregates. Based on the concentration dependencies, the number of peptide molecules in the aggregates was estimated. We find that this value ranges from 2 to 3, depending on the position of the spin label in the peptide sequence. The combined analysis of the distance spectra and the number of peptide molecules in the aggregates allows us to suggest that dimer formation is the prevailing mode of self-association. In the case of spin-labeled tylopeptin B, the molecules in the dimer are head-to-head oriented. In addition, the distance spectra of the aggregates show that the C-termini of the molecules in the tylopeptin B dimer are more mobile than the Ntermini. This phenomenon leads to an increase in the spread of the distances between the nitroxides as the label position is approaching the peptide C-terminus. For heptaibin, we show that two forms of dimerization (head-to-head and head-to-tail) occur. Finally, in addition to dimers, aggregates containing 3 or 4 peptide molecules, which give broad lines in the distance spectra, are seen in solution.     

LIGHT-INDUCED LEAKAGE OF SPIN LABEL MARKER FROM LIPOSOMES IN THE PRESENCE OF PHOTOTOXIC PHENOTHIAZINES

Abstract— Liposomes prepared from dipalmitoyl lecithin, cholesterol and dicetyl phosphate and containing a trapped spin label marker were exposed to long wavelength UV light in the presence of a series of phenothiazine tranquilizers. EPR spectroscopy was used to detect spin label marker released from liposomes, taking advantage of the disappearance of line broadening from electron spin exchange which occurred on spin label release. The minimum effective phototoxic dose in mice of these phenothiazines was also determined. Kinetic studies of light-induced spin label release from phenothiazine-sensitized liposomes showed that membrane damage was rapidly induced and that the damaging species were short-lived. The damage process was oxygen dependent and could be temporarily prevented by cysteamine or α-tocopherol added immediately before irradiation. Only those phenothiazines which mediated light-dependent liposomal membrane damage had phototoxic activity in mice and the degree of photosensitization was parallel in the two systems. In both photosensitization phenomena, the nature of the substituent at the phenothiazine 2-position was more important than the phenothiazine side chain.     

A Bifunctional Spin Label for Ligand Recognition on Surfaces

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Electron paramagnetic resonance (EPR) of biomolecules spin‐labeled with nitroxides can offer uniquely sensitive and selective insights into these processes, but new spin‐labeling strategies are needed. The synthesis and study of a bromoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported. The first examples of mannose and biotin ligands coupled to aqueous carboxy‐functionalized gold nanoparticles through a spin label are presented. EPR spectra were obtained for the spin‐labeled ligands both free in solution and attached to nanoparticles. The labels were recognized by the mannose‐binding lectin, Con A, and the biotin‐binding protein avidin‐peroxidase. Binding gave quantifiable changes in the EPR spectra from which binding profiles could be obtained that reflect the strength of binding in each case.