EPR Distance Measurements Research Articles

Gd(III)-Gd(III) EPR distance measurements--the range of accessible distances and the impact of zero field splitting.

Gd(III) complexes have emerged as spin labels for distance determination in biomolecules through double-electron-electron resonance (DEER) measurements at high fields. For data analysis, the standard approach developed for a pair of weakly coupled spins with S = 1/2 was applied, ignoring the actual properties of Gd(III) ions, i.e. S = 7/2 and ZFS (zero field splitting) ≠ 0. The present study reports on a careful investigation on the consequences of this approach, together with the range of distances accessible by DEER with Gd(III) complexes as spin labels. The experiments were performed on a series of specifically designed and synthesized Gd-rulers (Gd-PyMTA-spacer-Gd-PyMTA) covering Gd-Gd distances of 2-8 nm. These were dissolved in D2O-glycerol-d8 (0.03-0.10 mM solutions) which is the solvent used for the corresponding experiments on biomolecules. Q- and W-band DEER measurements, followed by data analysis using the standard data analysis approach, used for S = 1/2 pairs gave the distance-distribution curves, of which the absolute maxima agreed very well with the expected distances. However, in the case of the short distances of 2.1 and 2.9 nm, the distance distributions revealed additional peaks. These are a consequence of neglecting the pseudo-secular term in the dipolar Hamiltonian during the data analysis, as is outlined in a theoretical treatment. At distances of 3.4 nm and above, disregarding the pseudo-secular term leads to a broadening of a maximum of 0.4 nm of the distance-distribution curves at half height. Overall, the distances of up to 8.3 nm were determined, and the long evolution time of 16 μs at 10 K indicates that a distance of up to 9.4 nm can be accessed. A large distribution of the ZFS parameter, D, as is found for most Gd(III) complexes in a frozen solution, is crucial for the application of Gd(III) complexes as spin labels for distance determination via Gd(III)-Gd(III) DEER, especially for short distances. The larger ZFS of Gd-PyMTA, in comparison to that of Gd-DOTA, makes Gd-PyMTA a better label for short distances.

EPR Distance Measurements in Deuterated Proteins.

Pulsed electron double resonance technique, also known as double electron-electron resonance, jointly with site-directed spin labeling (SDSL) have been used extensively for studying structures and structural change. During the last decades, significant enhancements have been made by optimization of the experimental protocols, introducing new techniques for artifact suppression, and developing data analysis programs for extracting more reliable distance distributions. However, the distance determination by pulsed electron paramagnetic resonance is still facing some limitations, especially when studying spin-labeled proteins, due mainly to the fast relaxation time that imposes severe limitations on the maximum distances measurable and upon the sensitivity of such experiments. In the present work, we demonstrate the impact of the deuteration of the underlying protein, in addition to the solvent, on relaxation times, sensitivity, and on distance measurements.

Development and Application of Spin Traps, Spin Probes, and Spin Labels.

This chapter focuses on major achievements of the last decade in the synthesis and applications of spin traps, spin probes, and spin labels. Our discussion on spin trapping is mainly concerned with novel aspects of nitrones used as spin traps and with the kinetics caused by bioreductants. The second part of the chapter deals with recent developments in site-directed spin labeling (SDSL) for studying structure and functions of proteins and nucleic acids. We focus on SDSL EPR distance measurements using advanced trityl and nitroxide labels, on new approaches for incorporation of spin labels in biomolecules, and finally, on recent room/physiological temperature measurements made feasible by these novel spin labels.

Probing protein conformation in cells by EPR distance measurements using Gd3+ spin labeling.

Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron-electron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd(3+) chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells.

A modular approach for the synthesis of nanometer-sized polynitroxide multi-spin systems.

The synthesis of rigid symmetric polyradical model systems with inter-spin distances between 1.4 and 4 nm and their room temperature continuous wave (CW) EPR spectra are reported. Conditions for attachment of the spin-label via esterification have been optimized on the direct synthesis of polyradicals from commercially available polyphenols and the carboxylic acid functionalized nitroxide TPC. A common synthetic protocol utilizing 4-hydroxy-4'-iodobiphenyl as a key building block has been used to synthesize an equilateral biradical and a triradical in only two steps from commercially available starting materials. The first synthesis of a tetraradical based upon an adamantane core bearing six equivalent nitroxide-nitroxide distances is also reported. These systems are very promising candidates for studying multi-spin effects in pulsed EPR distance measurements.

Automated structure refinement for a protein heterodimer complex using limited EPR spectroscopic data and a rigid-body docking algorithm: a three-dimensional model for an ankyrin-CDB3 complex.

We report here specialized functions incorporated recently in the rigid-body docking software toolkit TagDock to utilize electron paramagnetic resonance derived (EPR-derived) interresidue distance measurements and spin-label accessibility data. The TagDock package extensions include a custom methanethiosulfonate spin label rotamer library to enable explicit, all-atom spin-label side-chain modeling and scripts to evaluate spin-label surface accessibility. These software enhancements enable us to better utilize the biophysical data routinely available from various spin-labeling experiments. To illustrate the power and utility of these tools, we report the refinement of an ankyrin:CDB3 complex model that exhibits much improved agreement with the EPR distance measurements, compared to model structures published previously.

Pulsed EPR distance measurements resolve the impact of site‐specific calmodulin methionine oxidation (1009.4)

We have examined the structural impact of oxidizing specific protein methionine (Met) residues in the C‐ter lobe of calmodulin (CaM); these oxidation sites are known to abolish CaM regulation of the major calcium release channel, the ryanodine receptor complex (RyR). We have linked oxidation‐induced changes in RyR regulation to changes in CaM‐RyR structure using (1) protein mutagenesis to mimic oxidation at specific sites and (2) spectroscopy to resolve oxidation‐induced changes in protein structural dynamics.Protein oxidation by reactive oxygen species (ROS) is strongly associated with loss of strength in skeletal muscle and is proposed to play major roles in aging and degenerative muscle disease. Targeted interventions that block or repair specific oxidative damage, while supporting normal levels of oxidation, could decrease morbidity associated with muscle dysfunction_this level of discrimination demands molecular‐level insights into the mechanisms that underlie the decline in muscle force associated with oxidative stress.Pulsed EPR distance measurements across CaM’s lobes were sensitive to large‐scale conformational changes that accompany both calcium binding and RyR peptide binding. In the absence of calcium, CaM was highly disordered, populating a broad distribution of conformations. Calcium binding strongly stabilized an elongated conformation, while RyR peptide binding to calcium‐loaded CaM strongly stabilized the compact conformation. CaM conformation, particularly the distribution over structural states, was sensitive to Met to Gln substitutions (M109Q and M124Q) designed to mimic CaM Met oxidation. Structural sensitivity to M‐to‐Q mutations was observed in both the presence and absence of calcium, and in complex with RyR peptide. We conclude that Met oxidation alters CaM’s functional interaction with RyR through changes in the orientation and flexibility of CaM’s lobes.Grant Funding Source: This work is supported by a University of Wisconsin‐La Crosse Faculty Research Grant to JC Klein, NI

Pulsed EPR Distance Measurements Resolve the Impact of Site-Specific Calmodulin Methionine Oxidation

We have examined the structural impact of oxidizing specific protein methionines (Met) in the C-ter lobe of calmodulin (CaM); these oxidation sites are known to abolish CaM regulation of the major calcium release channel, the ryanodine receptor complex (RyR). Protein oxidation by reactive oxygen species (ROS) is strongly associated with loss of strength in skeletal muscle and is proposed to play major roles in aging and degenerative muscle disease.We have linked oxidation-induced changes in RyR regulation to changes in CaM-RyR structure using (1) protein mutagenesis to mimic oxidation at specific sites and (2) spectroscopy to resolve oxidation-induced changes in protein structural dynamics. Pulsed EPR distance measurements across CaM's lobes (multiple pairs of labeling sites, one label on each lobe) were sensitive to large-scale conformational changes that accompany both calcium binding and RyR peptide binding. In the absence of calcium, CaM was highly disordered, populating a broad distribution of conformations. Calcium binding strongly stabilized the elongated conformation, while RyR peptide binding to calcium-loaded CaM strongly stabilized the compact conformation. CaM conformation, particularly the distribution over structural states, was sensitive to Met to Gln substitutions (M109Q and M124Q) designed to mimic CaM Met oxidation. Structural sensitivity to M-to-Q mutations was observed in both the presence and absence of calcium, and in complex with RyR peptide. We conclude that Met oxidation alters CaM's functional interaction with RyR through changes in the orientation and flexibility of CaM's lobes.This work is supported by a University of Wisconsin-La Crosse Faculty Research Grant to JC Klein, NIH grants to DD Thomas (R37AG026160 and P30AR057220), RJ Moen (F31AG037303), the Minnesota Biophysical Spectroscopy Center and the Minnesota Supercomputing Institute.

Biophysical Analysis of the Putative Heart Failure Drug Omecamtiv Mecarbil

The small molecule omecamtiv mecarbil (OM), currently in late phase-2 clinical trials for treatment of heart failure, activates force generation by cardiac myosin. How OM increases force generation is not known. Crosslinking and molecular docking studies suggest that the compound binds to the relay/SH1/converter region of myosin, hypothesized to control the actin-activated pre to post power-stroke structural transition in the myosin catalytic and light-chain domains. These transitions drive force production by myosin. We are currently using time-resolved FRET and EPR distance measurements to detect the pre- and post-power-stroke structural states of myosin. We have performed these studies on tissue-purified β-cardiac and α-skeletal myosin, and on a recombinant cys-lite myosin II catalytic domain model from Dictyostelium discoideum. In each of the myosin isoforms, we find that OM binding increases the mole fraction of the myosin post power-stroke structural state in the presence of ATP analogs. Transient time-resolved FRET experiments, mixing FRET-labeled myosin with ATP, show that drug binding also alters the structural kinetics of the myosin recovery-stroke. Ongoing studies are investigating the affect of OM on the actin-activated power-stroke. These results show that OM has a direct effect on the power-stroke structural transition that drives force generation in muscle, thus providing structural insight into the mechanism of this new potential therapy for heart failure. Our results also highlight the utility of structure-based time-resolved FRET and EPR assays for the discovery and characterization of allosteric enzyme modulators.

Long‐Range Distances in Amyloid Fibrils of α‐Synuclein from PELDOR Spectroscopy

Distance measurements: Pulsed EPR distance measurements combined with strategic spin labeling provide structural constraints at the molecular level for the fold of α-synuclein in amyloid fibrils (see picture; r=distance). The detection of interstrand distances in fibrils will potentially make it possible to extend these measurements to oligomeric states of these protein families.

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

Extending the distance range accessed with continuous wave EPR with Gd3+ spin probes at high magnetic fields.

Interspin distances between 0.8 nm and 2.0 nm can be measured through the dipolar broadening of the continuous wave (cw) EPR spectrum of nitroxide spin labels at X-band (9.4 GHz, 0.35 T). We introduce Gd(3+) as a promising alternative spin label for distance measurements by cw EPR above 7 Tesla, where the |-1/2〉 to |1/2〉 transition narrows below 1 mT and becomes extremely sensitive to dipolar broadening. To estimate the distance limits of cw EPR with Gd(3+), we have measured spectra of frozen solutions of GdCl3 at 8.6 T (240 GHz) and 10 K at concentrations ranging from 50 mM to 0.1 mM, covering a range of average interspin distances. These experiments show substantial dipolar broadening at distances where line broadening cannot be observed with nitroxides at X-band. This data, and its agreement with calculated dipolar-broadened lineshapes, show Gd(3+) to be sensitive to distances as long as ∼3.8 nm. Further, the linewidth of a bis-Gd(3+) complex with a flexible ∼1.6 nm bridge is strongly broadened as compared to the mono-Gd(3+) complex, demonstrating the potential for application to pairwise distances. Gd-DOTA-based chelates that can be functionalized to protein surfaces display linewidths narrower than aqueous GdCl3, implying they should be even more sensitive to dipolar broadening. Therefore, we suggest that the combination of tailored Gd(3+) labels and high magnetic fields can extend the longest interspin distances measurable by cw EPR from 2.0 nm to 3.8 nm. cw EPR data at 260 K demonstrate that the line broadening remains clear out to similar average interspin distances, offering Gd(3+) probes as promising distance rulers at temperatures higher than possible with conventional pulsed EPR distance measurements.

Characterization of Protein Conformational Changes with Sparse Spin-Label Distance Constraints.

The combination of site-directed spin labeling with pulse EPR distance measurements can provide a moderate number of distance constraints on the nanometer length scale for proteins in different states. By adapting an existing algorithm (Zheng, W.; Brooks, B. R. Biophys. J. 2006, 90, 4327) to the problem, we address the question to what extent conformational change can be characterized when the protein structure is known for one of the states, whereas only a sparse set of distance constraints between spin labels is available for the other state. We find that the type and general direction of the conformational change can be recognized, while the amplitude may be uncertain.

Heterooligomerization of Membrane Peptides Involved in Tuberculosis: MgtC and MgtR

Recent results have converged to highlight the role of hydrophobic peptides that form a novel class of active molecules in Escherichia coli and Salmonella enterica serovar Typhimurium. These peptides are apparently able to interact with membrane proteins leading to their inactivation through an unfolding process. MgtR, a highly hydrophobic 30 amino acid peptide, expressed in Salmonella typhimurium is one of these inactivating molecules. It has been shown that within the macrophage, the interaction between MgtR and the membrane protein MgtC leads to a decrease of Salmonella replication rate. This phenomenon can be explained by the importance of MgtC for the survival of the bacterium at low magnesium concentrations.To our knowledge, an interaction between two different peptides (heterooligomer) has never been studied in a membrane environment by NMR. Relying on the sequence homology between Salmonella typhimurium MgtC and Mycobacterium tuberculosis MgtC, the interaction between MgtR from Salmonella typhimurium and the transmembrane helix #4 of Mycobacterium tuberculosis MgtC is studied.Beyond the challenge consisting of solving a membrane protein structure, Solid state NMR and EPR are two powerful techniques to decipher protein dynamics and structure within the membrane hydrophobic core. Understanding the molecular mechanism of these peptide-protein interactions would offer great opportunities in pharmacology and drug design.Structural studies using solid state NMR spectroscopy of liposomal preparations for magic angle spinning experiments and oriented bilayer samples for additional structural restraints have been performed. EPR distance measurements were also performed on theses peptides in the membrane environment. The use of the NMR and EPR restrains combined, allowed us to propose a hypothetical interaction between these peptides in the membrane core.

Analysis of Conformational Changes in the DNA Junction-Resolving Enzyme T7 Endonuclease I on Binding a Four-Way Junction Using EPR

The four-way (Holliday) DNA junction is the central intermediate in homologous recombination. It is ultimately resolved into two nicked-duplex species by the action of a junction-resolving enzyme. These enzymes are highly selective for the structure of branched DNA, yet as a class these proteins impose significant distortion on their target junctions. Bacteriophage T7 endonuclease I selectively binds and cleaves DNA four-way junctions. The protein is an extremely stable dimer, comprising two globular domains joined by a β-strand bridge with each active site including amino acids from both polypeptides. The crystal structure of endonuclease I has been solved both as free protein and in complex with a DNA junction, showing that the protein, as well as the junction, becomes distorted on binding. We have therefore used site-specific spin-labeling in conjunction with EPR distance measurements to analyze induced fit in the binding of endonuclease I to a DNA four-way junction. The results support the change in protein structure as it binds to the junction. In addition, we have examined the structure of wild type and catalytically inactive mutants alone and in complex with DNA. We demonstrate the presence of hitherto undefined metastable conformational states within endonuclease I, showing how these states can be influenced by DNA-junction binding or mutations within the active sites. In addition, we demonstrate a previously unobserved instability in the N-terminal α1-helix upon active site mutation. These studies reveal that structural changes in both DNA and protein occur in the action of this junction-resolving enzyme.

Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans . It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = (5)/(2) high-spin (HS) and Fe(III) S = (1)/(2) low-spin (LS) hemes. (14)N ENDOR spectroscopy on the S = (5)/(2) signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = (5)/(2) heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T(1)-spin-lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.

W-Band pulse EPR distance measurements in peptides using Gd3+–dipicolinic acid derivatives as spin labels

We present high field DEER (double electron-electron resonance) distance measurements using Gd(3+) (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd(3+) spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd(3+) was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd(3+) labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd(3+) labeled peptide we explored the effect of the solution molar ratio of Gd(3+) and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd(3+)]/[Tag] ratio is crucial because excess Gd(3+) masked the DEER effect and too little Gd(3+) resulted in the formation of Gd(3+)-tag(2) complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd(3+) concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd(3+) ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.

EPR distance measurements in deuterated proteins

One of the major problems facing distance determination by pulsed EPR, on spin-labeled proteins, has been the short relaxation time Tm. Solvent deuteration has previously been used to slow relaxation and so extend the range of distance measurement and sensitivity. We demonstrate here that deuteration of the underlying protein, as well as the solvent, extends the Tm to a considerable degree. Longer Tm gives greatly enhanced sensitivity, much extended distance measurement, more reliable distance distribution calculation and better baseline correction.

Structural Dynamics Of The Actin Binding Cleft Of Dictyostelium Myosin II Analyzed By Stopped Flow Time-Resolved FRET

Using site-directed fluorescence labeling, transient time resolved FRET ([TR]2FRET) and time resolved fluorescence anisotropy, we have measured actin- and nucleotide-induced structural changes within the actin binding cleft of Dictyostelium myosin II. In a recent report using time-resolved pulsed EPR spectroscopy, distances were measured between paramagnetic probes attached to the upper and lower 50 kDa subdomains of Dictyostelium myosin II (Klein et al., PNAS, 105:12867-72). These results support the hypothesis that the actin-binding cleft closes partially upon actin binding, but also suggested that both open and closed conformations are simultaneously present, with nucleotides and actin controlling the open-closed equilibrium. Due to technical constraints, those EPR distance measurements were limited to frozen samples. In the present study, time-resolved fluorescence is used to probe the cleft in solution under more physiologically relevant conditions, including the transient phase of the ATPase reaction. Fluorescent probes were attached to engineered Cys residues in the upper and lower 50 kDa subdomains and used to measure the distance across the cleft. Single probes attached to either subdomain were used in combination with fluorescent nucleotides to monitor the coupling between the actin-binding cleft and the active site. Here we use a combination of transient time resolved FRET and transient time resolved fluorescence anisotropy to probe the equilibrium between open and closed cleft conformations. In the key experiments, a complete nanosecond time-resolved fluorescence decay was measured, defining the detailed distance distribution between probes, every 0.1 ms following rapid mixing (stopped flow), thus yielding high-resolution structural information on the sub-millisecond time scale. The results provide new insights into the coupling between the actin-binding cleft, the active site, and actin binding.

Gd3+ Complexes as Potential Spin Labels for High Field Pulsed EPR Distance Measurements

Pulse electron−electron double resonance distance measurements between two high spin Gd3+ ions in a novel bis-Gd3+ complex involving two pyridine-based gadolinium tetracarboxylate systems linked by a rigid aryl−alkyne unit were carried out at Ka- and W-band frequencies. The experimental distance found was 2.02 ± 0.02 nm, and it was further compared with the Gd3+−Gd3+ distance (2.2126 nm) determined by density functional theory.