Nitroxide Labels Research Articles

Ionizable Nitroxides for Studying Local Electrostatic Properties of Lipid Bilayers and Protein Systems by EPR.

Electrostatic interactions are known to play a major role in the myriad of biochemical and biophysical processes. Here, we describe biophysical methods to probe local electrostatic potentials of proteins and lipid bilayer systems that are based on an observation of reversible protonation of nitroxides by electron paramagnetic resonance (EPR). Two types of probes are described: (1) methanethiosulfonate derivatives of protonatable nitroxides for highly specific covalent modification of the cysteine's sulfhydryl groups and (2) spin-labeled phospholipids with a protonatable nitroxide tethered to the polar head group. The probes of both types report on their ionization state through changes in magnetic parameters and degree of rotational averaging, thus, allowing the electrostatic contribution to the interfacial pKa of the nitroxide, and, therefore, the local electrostatic potential to be determined. Due to their small molecular volume, these probes cause a minimal perturbation to the protein or lipid system. Covalent attachment secures the position of the reporter nitroxides. Experimental procedures to characterize and calibrate these probes by EPR, and also the methods to analyze the EPR spectra by simulations are outlined. The ionizable nitroxide labels and the nitroxide-labeled phospholipids described so far cover an exceptionally wide range of ca. 2.5-7.0 pH units, making them suitable to study a broad range of biophysical phenomena, especially at the negatively charged lipid bilayer surfaces. The rationale for selecting proper electrostatically neutral interface for probe calibration, and examples of lipid bilayer surface potential studies, are also described.

Gd³⁺ Spin Labeling for Measuring Distances in Biomacromolecules: Why and How?

Applications of distance measurements by pulse dipolar electron-paramagnetic resonance (PD-EPR) spectroscopy to structural biology are based on introducing spin labels (SLs) at well-defined locations in the biomacromolecule. The most commonly used SLs are nitroxyl radicals, but recently SLs based on high-spin Gd(3+) (S=7/2) complexes have been shown to be an attractive alternative for PD-EPR, particularly double electron-electron resonance (DEER), at spectrometer frequencies higher than 30 GHz. In this chapter, we describe the advantage of using this new family of SLs in terms of sensitivity, stability, and chemical diversity. We present current labeling strategies for proteins, discuss the approximations under which DEER data analysis of a pair of Gd(3+) SLs (GdSLs) is equivalent to that of a pair of S=1/2 SLs, and discuss the reduction in multispin effects in a cluster of GdSLs, as opposed to a cluster of nitroxide labels, which can be found in oligomeric systems. In addition, we provide a brief overview of the current, rather limited, knowledge of Gd(3+) phase relaxation behavior and describe experimental strategies in terms of optimizing sensitivity. The possibility of using several types of SLs in a system allows one to isolate effects due to the chemical nature of the SL itself; several such examples are presented, focusing on comparing nitroxide and GdSLs. Finally, we will discuss the initial results on in-cell DEER with GdSLs.

Pulsed EPR spectroscopy distance measurements of DNA internally labelled with Gd(3+)-DOTA.

Gd(3+) is increasingly used in EPR spectroscopy due to its increased intracellular stability and signal-to-noise ratios. Here we present the incorporation of Gd(3+)-DOTA into internal positions in DNA. Distance measurements via pulsed Electron Paramagnetic Resonance (EPR) spectroscopy in vitro and in cellula proved enhanced stability and efficiency compared to nitroxide labels.

Site directed nitroxide spin labeling of oligonucleotides for NMR and EPR studies

Nitroxide labels are useful probes of biomolecule structure that can be detected by NMR and EPR spectroscopy. Although many methods exist for labeling oligonucleotides with nitroxides, most require reagents that are expensive or laborious to prepare. A simpler approach is described herein using commercially available phosphorothioate oligonucleotides and 2-(3-iodoacetamidomethyl)-PROXYL. We describe semi-optimized conditions for labeling DNA and RNA oligonucleotides and methodology for purifying and identifying these reagents by MALDI-MS. The nitroxide label showed some propensity to hydrolyze at high temperature and over prolonged periods at room temperature. Nitroxide-labeled DNA oligonucleotides gave characteristic EPR spectra and caused the disappearance of bound protein signals in 15N HSQC spectra consistent with the paramagnetic relaxation enhancement (PRE) effect.

Diversification of EPR signatures in site directed spin labeling using a β-phosphorylated nitroxide

Site Directed Spin Labeling (SDSL) combined with EPR spectroscopy is a very powerful approach to investigate structural transitions in proteins in particular flexible or even disordered ones. Conventional spin labels are based on nitroxide derivatives leading to classical 3-line spectra whose spectral shapes are indicative of the environment of the labels and thus constitute good reporters of structural modifications. However, the similarity of these spectral shapes precludes probing two regions of a protein or two partner proteins simultaneously. To overcome the limitation due to the weak diversity of nitroxide label EPR spectral shapes, we designed a new spin label based on a β-phosphorylated nitroxide giving 6-line spectra. This paper describes the synthesis of this new spin label, its grafting at four different positions of a model disordered protein able to undergo an induced α-helical folding and its characterization by EPR spectroscopy. For comparative purposes, a classical nitroxide has been grafted at the same positions of the model protein. The ability of the new label to report on structural transitions was evaluated by analyzing the spectral shape modifications induced either by the presence of a secondary structure stabilizer (trifluoroethanol) or by the presence of a partner protein. Taken together the results demonstrate that the new phosphorylated label gives a very distinguishable signature which is able to report from subtle to larger structural transitions, as efficiently as the classical spin label. As a complementary approach, molecular dynamics (MD) calculations were performed to gain further insights into the binding process between the labeled NTAIL and PXD. MD calculations revealed that the new label does not disturb the interaction between the two partner proteins and reinforced the conclusion on its ability to probe different local environments in a protein. Taken together this study represents an important step forward in the extension of the panoply of SDSL-EPR approaches.

Gadolinium(III) Spin Labels for High‐Sensitivity Distance Measurements in Transmembrane Helices

Distance determination, by pulse EPR techniques, between two spin labels attached to biomolecules has become an attractive methodology to probe conformations and assemblies of biomolecules in frozen solutions. Among these techniques, double electron-electron resonance (DEER or PELDOR), which can access distances in the range of 1.7 to 8 nm, is highly popular, and the most widely used spin labels are nitroxide radicals. Membrane proteins in their natural environment are of particular interest for DEER applications, since those pose a considerable challenge for Xray crystallography and NMR spectroscopy. DEER studies of peptides and proteins in either reconstituted or model membranes are considerably more challenging than those in solution, because the high local concentration of the spins in the membrane decreases the phase memory time and, therefore, sensitivity. Most DEER measurements on nitroxide-labeled biomolecules are carried out at X-band frequencies (9.5 GHz, 0.35 T), and recently such measurements were demonstrated in frozen cells. A major difficulty of such measurements is the reduction of nitroxides in the cell, which severely limits the scope of such exciting developments. Recently, Gd (S= 7/2) spin labels have been suggested as an alternative to nitroxide spin labels for W-band and Qband DEER distance measurements. Gd tags can be attached to proteins, similar to nitroxides, by site-directed spin labeling (SDSL). Gd features high sensitivity at high frequencies and the DEER measurements are free of orientation selection effects so that the distance distribution can readily be extracted from a single DEER measurement. Moreover, in the context of future development of in-cell DEER measurements, Gd chelates are stable under in vivo conditions as known from their applications as contrast agents for magnetic resonance imaging (MRI). Gd-Gd DEER has been demonstrated on model systems, proteins, peptides, and DNA, all in isotropic membrane-free solutions. Distance measurements between a Gd label and a nitroxide label have also been shown to yield attractive sensitivity. In this work, we continue to develop the approach of Gd–Gd DEER distance measurements and demonstrate for the first time such measurements in a model membrane. The model system we chose consists of the well-studied transmembrane helical WALP peptides in 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) vesicles. We demonstrate the sensitivity of W-band Gd–Gd DEER to small distance variations in a membrane. Using WALP peptides of different lengths we show that such measurements pick up, in addition to the helix extension, also subtle “cis–trans” effects arising from different positions of the labels with respect to the helix axes. In addition, we report the effect of the spin label interaction with the membranes on the measured distance distribution. We compared W-band DEER on WALP23 labeled with two different Gd tags with X-band DEER onWALP23 labeled with nitroxide tags. Here we used X-band, rather than W-band, to avoid complications owing to orientation selection. The differences observed are important and suggest that by employing different spin labels such effects can be isolated. Finally, we show that the effect of hydrophobic mismatch between peptide and membrane can be explored by Gd–Gd DEER. WALP23 was labeled at the N and C termini (see Table 1) with two nitroxides (WAL23-NO) using (l-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSSL) and two different Gd-DOTA derivatives, shown in Figure 1. WALP23-DOTA is labeled with a DOTA chelate and WALP23-C1 with DOTA with phenylethylamine substituents. The bulky substituents were designed to restrict the flexibility of the tag. The hydrophobic length of WALP23 (ca. 2.6 nm) is very close to that of the hydrophobic thickness of DOPC bilayers (ca. 2.7 nm). The sample composition was 50 mm WALP23 in DOPC multilamellar vesicles (MLV; 1:1000 peptide/lipid molar ratio). Details of the sample preparation are given in the Supporting Information. [*] Dr. E. Matalon, Dr. A. Feintuch, Prof. D. Goldfarb Department of Chemical Physics, Weizmann Institute of Science Rehovot, 76100 (Israel) E-mail: Daniella.goldfarb@weizmann.ac.il

Distance determination between low-spin ferric haem and nitroxide spin label using DEER: the neuroglobin case

This work demonstrates for the first time the feasibility of using double electron–electron resonance (DEER) to determine the inter-spin distance between nitroxide spin labels and low-spin (S = 1/2) ferric haem centres. For these means, two human neuroglobin variants were spin labelled leading to singly labelled haem proteins with the nitroxide label on one of the natural Cys residues (Cys55 or Cys120). Room-temperature electron paramagnetic resonance was used to characterise the mobility of the nitroxide labels and X- and Q-band DEER experiments were performed to detect nitroxide–haem distances. Effects of residual nuclear modulations in the DEER traces were carefully evaluated. The DEER-derived distances were compared with theoretical predictions from an X-ray diffraction structure of human neuroglobin using a rotamer library approach as well as with distance information obtained from electron relaxation measurements. The structural biological implications of the spin-labelled side chains’ dynamics and of the obtained distances are also discussed.

ESR Study of Interfacial Hydration Layers of Polypeptides in Water-Filled Nanochannels and in Vitrified Bulk Solvents

There is considerable evidence for the essential role of surface water in protein function and structure. However, it is unclear to what extent the hydration water and protein are coupled and interact with each other. Here, we show by ESR experiments (cw, DEER, ESEEM, and ESE techniques) with spin-labeling and nanoconfinement techniques that the vitrified hydration layers can be evidently recognized in the ESR spectra, providing nanoscale understanding for the biological interfacial water. Two peptides of different secondary structures and lengths are studied in vitrified bulk solvents and in water-filled nanochannels of different pore diameter (6.1∼7.6 nm). The existence of surface hydration and bulk shells are demonstrated. Water in the immediate vicinity of the nitroxide label (within the van der Waals contacts, ∼0.35 nm) at the water-peptide interface is verified to be non-crystalline at 50 K, and the water accessibility changes little with the nanochannel dimension. Nevertheless, this water accessibility for the nanochannel cases is only half the value for the bulk solvent, even though the peptide structures remain largely the same as those immersed in the bulk solvents. On the other hand, the hydration density in the range of ∼2 nm from the nitroxide spin increases substantially with decreasing pore size, as the density for the largest pore size (7.6 nm) is comparable to that for the bulk solvent. The results demonstrate that while the peptides are confined but structurally unaltered in the nanochannels, their surrounding water exhibits density heterogeneity along the peptide surface normal. The causes and implications, especially those involving the interactions between the first hydration water and peptides, of these observations are discussed. Spin-label ESR techniques are proven useful for studying the structure and influences of interfacial hydration.

Restrained-Ensemble Molecular Dynamics Simulations Based on Distance Histograms from Double Electron–Electron Resonance Spectroscopy

DEER (double electron-electron resonance) spectroscopy is a powerful pulsed ESR (electron spin resonance) technique allowing the determination of spin-spin distance histograms between site-directed nitroxide label sites on a protein in their native environment. However, incorporating ESR/DEER data in structural refinement is challenging because the information from the large number of distance histograms is complex and highly coupled. Here, a novel restrained-ensemble molecular dynamics simulation method is developed to incorporate the information from multiple ESR/DEER distance histograms simultaneously. Illustrative tests on three coupled spin-labels inserted in T4 lysozyme show that the method efficiently imposes the experimental distance distribution in this system. Different rotameric states of the χ1 and χ2 dihedrals in the spin-labels are also explored by restrained ensemble simulations. Using this method, it is hoped that experimental restraints from ESR/DEER experiments can be used to refine structural properties of biological systems.

ChemInform Abstract: Determining the Orientation and Localization of Membrane‐Bound Peptides

AbstractReview: 121 refs.

Rotamer Library of Spin Labeled Cysteines Attached to T4 Lysozyme Deduced from Molecular Dynamics Simulations Constrained by Double Electron-Electron Resonance (Deer) Experiments

DEER (Double Electron Electron Resonance) is a powerful pulsed EPR technique allowing the determination of spin-spin distance histograms between side-directed nitroxide label sites on a protein in their native environment. In this study, a novel molecular dynamics simulation method, the restrained ensemble (RE) method, has been used for the refinement of spin labels inserted in T4 Lysozyme (T4L) structures. In the RE method, the spin-spin distance distribution histograms calculated from a multiple-copy molecular dynamics simulations are forced, via a special ensemble-based energy restraint, to match those extracted from 51 EPR/DEER experimental spin-spin pairs. To examine the effect of the restraint on the rotameric state of the spin label at the 37 different sites in T4L, conventional long molecular dynamics (MD) and locally enhanced sampling (LES) simulations were also performed. As expected, the distance histograms obtained from the RE simulations are very similar to those obtained from experiment; however, the distance histograms from conventional long MD and LES simulations deviate from experiment. From the analysis of these simulations, a general trend is found in rotamer population distribution along five dihedral angles connecting the nitroxide ring to the protein backbone. Overall, the rotamer population obtained from the RE simulation agrees with those obtained from x-ray structure. From the analysis of the dynamics of nitroxide atoms in spin label side chains, a force field for a pseudo nitroxide dummy atom has been parameterized. This pseudo nitroxide was used in an RE simulation coupled with the EPR/DEER experimental distance distribution data to refine distorted structures of T4L. Thus, experimental restraints from DEER experiments in conjunction with RE simulations can be used to refine structural propertied of biological systems.

Ultrafast Red Light Activation of Synechocystis Phytochrome Cph1 Triggers Major Structural Change to Form the Pfr Signalling-Competent State

Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel ‘head-to-head’ arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.

Dynamics and Binding Affinity of Spin-Labeled Stearic Acids in β-Lactoglobulin: Evidences from EPR Spectroscopy and Molecular Dynamics Simulation

β-Lactoglobulin (β-LG) is a member of the lipocalin protein family involved in the transport of fatty acids and other small hydrophobic molecules. The main binding site is at a central cavity, referred to as "calyx", formed by the protein β-barrel sandwich. Continuous-wave and pulsed Fourier transform electron spin resonance (cw- and FT-EPR) spectroscopy and molecular dynamics (MD) simulation were combined to investigate the interaction of fatty acids with bovine β-LG. Stearic acid bearing the nitroxide label at different positions, n, along the acyl chain (n-SASL, n = 5, 7, 10, 12, 16) were used. The EPR data show that the protein affinity for SASL decreases on going from n = 5 to 16. This behavior is due to the accommodation of the SASL in the protein calyx, which is hampered by steric hindrance of the doxyl ring for n ≥ 10, as evidenced by MD data. Conformation and dynamics of 5-SASL are similar to those of the unlabeled stearate molecule. 5-SASL in the protein binding site undergoes librational motion of small amplitude on the nanosecond time scale at cryogenic temperature and rotational dynamics with correlation time of 4.2 ns at physiological temperature. The results highlight the dynamical features of fatty acids/β-LG interaction.

Determining the orientation and localization of membrane-bound peptides.

Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance.

EPR Study of Spin Labeled Brush Polymers in Organic Solvents

Spin-labeled polylactide brush polymers were synthesized via ring-opening metathesis polymerization (ROMP), and nitroxide radicals were incorporated at three different locations of brush polymers: the end and the middle of the backbone, and the end of the side chains (periphery). Electron paramagnetic resonance (EPR) was used to quantitatively probe the macromolecular structure of brush polymers in dilute solutions. The peripheral spin-labels showed significantly higher mobility than the backbone labels, and in dimethylsulfoxide (DMSO), the backbone end labels were shown to be more mobile than the middle labels. Reduction of the nitroxide labels by a polymeric reductant revealed location-dependent reactivity of the nitroxide labels: peripheral nitroxides were much more reactive than the backbone nitroxides. In contrast, almost no difference was observed when a small molecule reductant was used. These results reveal that the dense side chains of brush polymers significantly reduce the interaction of the backbone region with external macromolecules, but allow free diffusion of small molecules.

The Dynamic Structure of Filamentous Tau

Filaments of the protein tau are a characteristic occurrence in Alzheimer disease and many other neurodegenerative disorders and the distribution of tau filaments correlates well with the loss of neurons and cognitive functions in Alzheimer disease. Filament formation of tau filaments is based on structural transitions from random coil to b-structure, to give the paired helical filaments (PHFs) which share common characteristics of amyloid fibrils. Protease digestion and solvent-accessibility studies demonstrated that the “core” of PHFs is mainly built from repeat sequences in the C-terminal half of the tau protein. The PHF core is surrounded by a “fuzzy coat”, of more than 200 residues that come from the N-terminal half of the protein as well as the C-terminus (Figure 1a). Electron paramagnetic resonance and nuclear magnetic resonance (NMR) suggested that residues within the fuzzy coat are highly flexible. Biochemical studies have shown that the fuzzy coat is important for tau aggregation as well as neurotoxicity. Herein we characterized the dynamic structure of PHFs formed by 441-residue tau (htau40), the longest isoform of tau present in the human central nervous system (Figure 1a), at single-residue level using NMR spectroscopy. We aggregated N-labeled htau40 into insoluble filaments. NMR diffusion experiments demonstrated that the observed NMR signals arises from aggregated tau protein with a molecular mass of more than 1 MDa (Figure 1b). In a two-dimensional heteronuclear single quantum coherence (HSQC) spectrum employing high-resolution magic-angle spinning (HR-MAS) (see Figure S1 in the Supporting Information), we observed about 260 signals (Figure 1c and Figure S2 and S3 in the Supporting Information). Sequencespecific resonance assignment of 244 of these signals (BMRB accession number: 17920; see Figure S2 in the Supporting Information) identified most of the residues in the N-terminal domain up to Thr212 and at the C-terminus starting at Val399. No signals were detected for residues between Thr212 and Val399, suggesting that residues in the central domain are too immobile to be detected by liquid-state NMR spectroscopy in agreement with previous studies. Comparison with monomeric htau40 revealed that the NMR resonances of many residues were strongly reduced in filamentous htau40 (Figure 1d,e). Most strikingly, the sections His121– Lys130 and Met1–Gly37 that are separated from the fibril core by 170 residues or more, showed changes in position and intensity of NMR signals (Figure 1e and Figure S2c in the Supporting Information). The presence of chemical exchange in these regions was further supported by N spin relaxation measurements (Figure 1 f and Supporting Figure S3). In agreement with chemical exchange, additional peaks were observed in close proximity to several of these residues (Figure 1 f and Figure S4 in the Supporting Information). The additional signals could not be connected in triple-resonance experiments or using exchange spectroscopy because of low signal-to-noise and signal overlap. Therefore, we assigned the additional peaks to the residue for which the assigned crosspeak of the major peak set had the greatest similarity in chemical shifts and paramagnetic relaxation enhancement (see Figure S5 in the Supporting Information). This procedure indicates that the additional peaks arise from residues at the Nand C-terminus. No peak doubling was observed at the Nand C-terminus in monomeric tau (see Figure S4 in the Supporting Information), highlighting the specificity of the multiple conformations in PHF tau. We revealed the identity of the PHF-specific conformations through measurements of paramagnetic relaxation enhancements (PREs), in which nitroxide spin labels are attached to cysteine residues at various positions in the PHF tau. The resulting broadening of amide resonances caused by enhanced relaxations rate through the paramagnetic nitroxide label, is quantified through the intensity ratios in the paramagnetic and diamagnetic states (Figure 2). The PRE effect scales as the inverse sixth power of the distance between the unpaired electron of the nitroxide unit and the NMR spin, providing a powerful probe of distances. N spin relaxation times (Figure 1 f) indicate that the fuzzy coat of PHFs is highly dynamic on a broad scale suggesting that the correlation time of the electron—amide proton internuclear vector is comparable to that of small water soluble proteins. Initially, we measured PRE broadening for PHFs with a [*] S. Bibow, Dr. M. D. Mukrasch, Prof. Dr. C. Griesinger, Prof. Dr. M. Zweckstetter Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 Gcttingen (Germany) E-mail: mzwecks@gwdg.de

High-Field Dipolar Electron Paramagnetic Resonance (EPR) Spectroscopy of Nitroxide Biradicals for Determining Three-Dimensional Structures of Biomacromolecules in Disordered Solids

We consider the state-of-the-art capabilities and future perspectives of electron-spin triangulation by high-field/high-frequency dipolar electron paramagnetic resonance (EPR) techniques designed for determining the three-dimensional structure of large supra-molecular complexes dissolved in disordered solids. These techniques combine double site-directed spin labeling (SDSL) with orientation-resolving pulsed electron-electron double resonance (PELDOR) spectroscopy. In particular, we appraise the prospects of angular triangulation, which extends the more familiar distance triangulation. As a model case for spin-labeled proteins, the three-dimensional structures of two nitroxide biradicals with rather stiff bridging blocks and deuterated nitroxide headgroups have been derived. To this end we applied 95 GHz high-field electron dipolar EPR spectroscopy with the microwave pulse-sequence configurations for PELDOR and relaxation-induced dipolar modulation enhancement (RIDME). Various specific spectroscopic strategies are discussed to overcome the problems of overlapping spectra of the chemically identical nitroxide labels when attached to macromolecular systems. We conclude that due to the high detection sensitivity and spectral resolution the combination of SDSL with high-field RIDME/PELDOR stands out as an extremely powerful tool for 3D structure determination of large disordered systems. The approach compares favorably with other structure-determining magnetic-resonance methods. This holds true both for stable and transient radical-pair states. Angular constraints are provided in addition to distance constraints obtained for the same sample. Thereby, the number of necessary distance constraints is strongly reduced. Since each measurement of a distance constraint requires an additional doubly spin-labeled sample, the reduction of necessary distance constraints is another appealing aspect of orientation-resolving EPR spin triangulation for protein structure determination.

Pulsed dipolar spectroscopy distance measurements in biomacromolecules labeled with Gd(III) markers

This work demonstrates the feasibility of using Gd(III) tags for long-range Double Electron Electron Resonance (DEER) distance measurements in biomacromolecules. Double-stranded 14- base pair Gd(III)-DNA conjugates were synthesized and investigated at K a band. For the longest Gd(III) tag the average distance and average deviation between Gd(III) ions determined from the DEER time domains was about 59 ± 12 Å. This result demonstrates that DEER measurements with Gd(III) tags can be routinely carried out for distances of at least 60 Å, and analysis indicates that distance measurements up to 100 Å are possible. Compared with commonly used nitroxide labels, Gd(III)-based labels will be most beneficial for the detection of distance variations in large biomacromolecules, with an emphasis on large scale changes in shape or distance. Tracking the folding/unfolding and domain interactions of proteins and the conformational changes in DNA are examples of such applications.

Distance measurements in model bis-Gd(III) complexes with flexible “bridge”. Emulation of biological molecules having flexible structure with Gd(III) labels attached

In this work, we continue to explore Gd(III) as a possible spin label for high-field Double Electron–Electron Resonance (DEER) based distance measurements in biological molecules with flexible geometry. For this purpose, a bis-Gd(III) complex with a flexible “bridge” was used as a model. The distances in the model were expected to be distributed in the range of 5–26 Å, allowing us to probe the shortest limits of accessible distances which were found to be as small as 13 Å. The upper distance limit for these labels was also evaluated and was found to be about 60 Å. Various pulse duration setups can result in apparent differences in the distribution function derived from DEER kinetics due to short distance limit variations. The advantages, such as the ability to perform measurements at cryogenic temperatures and high repetition rates simultaneously, the use of very short pumping and observation pulses without mutual interference, the lack of orientational selectivity, as well as the shortcomings, such as the limited mw operational frequency range and intrinsically smaller amplitude of oscillation related to dipolar interaction as compared with nitroxide spin labels are discussed. Most probably the use of nitroxide and Gd-based labels for distance measurements will be complementary depending on the particulars of the problem and the availability of instrumentation.

Accurate Distance Constraints for RNA Structures Using Deer Spectroscopy

Understanding structure-function relationships in RNA and RNA-protein complexes requires robust methods for obtaining structural information on a variety of length scales. DEER (double electron-electron resonance) is emerging as a powerful method for very accurate (± 2 A over 15-80 A) distance measurements between pairs of nitroxide labels that can be placed using several available conjugation sites in RNA nucleobases or phosphodiester linkages. Here, we show the potential for DEER spectroscopy in monitoring global RNA folding and also small changes in RNA structure within a model system that is based on the Hammerhead ribozyme. This catalytically active RNA, a three-helix junction motif with a buried active site, undergoes cation-dependent folding transitions that are linked to activity. Nitroxide labels placed at strategic positions allow helix-docking and active-site core rearrangements to be monitored by measuring the dipolar coupling between paramagnetic sites. This poster will present the results of DEER measurements obtained at both X-band and Q-band, where the higher-frequency Q-band spectroscopy significantly enhances the sensitivity of this technique. Mg2+-dependent global folding, and evidence for a smaller local structural change with higher added metal concentrations, are both observed in this RNA. Since labels can be placed at targeted sites within both nucleic acids and proteins, and there is no inherent limitation on macromolecular size, DEER spectroscopy has potential for obtaining high-resolution structural information in complex RNAs and in large RNA or DNA-protein complexes.