Double Electron-electron Resonance Distance Research Articles

Guiding Double Electron Electron Resonance Experiments using Information Theory and Molecular Dynamics Simulations

Flexible proteins present a unique set of challenges for refinement with either computational or experimental techniques: because these proteins occupy a large conformational space, standard molecular dynamics (MD) simulations can be prohibitively expensive, and high-resolution experimental techniques like x-ray crystallography intentionally capture only small set of low energy states. A new approach is therefore needed to capture the conformational ensembles of flexible proteins. Double electron-electron resonance (DEER) spectroscopy, an experimental technique that measures distance distributions between pairs of spin-labeled residues on a protein, provides quantitative information about less populated but still potentially important conformational states. However, because DEER experiments are costly and time consuming, only a small subset of pairs can be measured, often chosen arbitrarily. If instead of selecting an arbitrary or random subset, we select a subset of pairs such that they provide the maximum amount of information about the conformational ensemble, DEER could become an even more powerful tool for refinement. We have therefore developed 1) a new information-theoretic metric that ranks pairs of residues based on how well they refine a conformational ensemble and 2) a new methodology which identifies a set of highly informative pairs that perform well under this metric. Specifically, we incorporate DEER distance distributions into restrained-ensemble MD simulations, utilize the data from these simulations to identify a set of maximally-informative and minimally-redundant pairs, measure the pairs with DEER, and demonstrate the use of these pairs in refining the conformational ensemble of a flexible protein.

Rotameric preferences of a protein spin label at edge‐strand β‐sheet sites

Protein spin labeling to yield the nitroxide-based R1 side chain is a powerful method to measure protein dynamics and structure by electron spin resonance. However, R1 measurements are complicated by the flexibility of the side chain. While analysis approaches for solvent-exposed α-helical environment have been developed to partially account for flexibility, similar work in β-sheets is lacking. The goal of this study is to provide the first essential steps for understanding the conformational preferences of R1 within edge β-strands using X-ray crystallography and double electron electron resonance (DEER) distance measurements. Crystal structures yielded seven rotamers for a non-hydrogen-bonded site and three rotamers for a hydrogen-bonded site. The observed rotamers indicate contextual differences in R1 conformational preferences compared to other solvent-exposed environments. For the DEER measurements, each strand site was paired with the same α-helical site elsewhere on the protein. The most probable distance observed by DEER is rationalized based on the rotamers observed in the crystal structure. Additionally, the appropriateness of common molecular modeling methods that account for R1 conformational preferences are assessed for the β-sheet environment. These results show that interpretation of R1 behavior in β-sheets is difficult and indicate further development is needed for these computational methods to correctly relate DEER distances to protein structure at edge β-strand sites.

Investigation of the Binding of the Human KCNE1 Protein with the Voltage Gated Potassium Channel KCNQ1 using DEER EPR Spectroscopy

Voltage-gated ion channels are essential for the electrical excitability of neurons, muscles and other excitable cells. KCNE1 is a single transmembrane protein that modulates the activity of voltage gated potassium ion channels (Kv). In the human heart, KCNE1 (E1) interacts with KCNQ1 (Q1) and decreases the rate of channel activation, increases conductance, and generates a slowly activating K+ current critical for cardiac repolarization. Mutations on either KCNE1 or KCNQ1 genes in E1/Q1 complex can lead to cardiac long QT syndrome. Despite the biological significance of the Q1/E1 interaction, its exact nature is not fully understood. In this study, we have used electron paramagnetic resonance (EPR) experiments and double electron electron resonance (DEER) distance measurements to provide the direct evidence of binding activities of KCNQ1 with KCNE1. CW-EPR titration experiment performed on different sites of the C-terminus of KCNQ1 indicated that the EPR lineshape broadening increases with the addition of WT-KCNE1 and saturates at the molar ratio of 1:3 (Q1:E1) suggesting that the spin label motion on Q1 decreases, while interacting with E1. DEER distance measurements on the doubly spin labeled Q1 (374/395 and 524/540) change while the distance measurement on the doubly spin labeled Q1 (445/460) remains the same upon addition of WT-KCNE1 indicating that the regions close to sites 374/395 and 524/540 are interacting strongly with KCNE1. DEER was also used to measure distances between the spin labels attached on E1 and Q1 separately in the Q1/E1 complex to determine the interacting sites in more detail. These results provide direct evidence of binding of Q1 with E1 and will be very useful for determining the structural model of the Q1/E1 complex.

Neisserial Opa Protein Loop Dynamics and Mechanism of Interaction with Host CEACAM Receptors

Neisseria gonorrhoeae and N. meningitides are human pathogens which infect millions each year and are becoming increasingly antibiotic resistant. These bacteria utilize opacity-associated (Opa) proteins to mediate bacterial uptake into non-phagocytic and phagocytic cells as a means of infection. Opa proteins primarily engage carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs), hijacking host cellular mechanisms to induce bacterial engulfment. The Opa family of proteins are outer membrane β-barrels with eight transmembrane strands and four extracellular loops. Opa proteins bind CEACAMs with high affinity and specificity, yet the loops important to receptor engagement are highly diverse in sequence among Opa variants. We aim to determine how sequence diversity in the receptor-interacting domains of Opa loops facilitates engagement of CEACAM receptors. Results from CW-EPR and NMR relaxation experiments indicate the Opa loops are dynamic on the nanosecond timescale in lipid and detergent environments. Such flexibility may enable sampling of transient structures and interactions that promote receptor engagement. To determine if the conformation of the bound loops is sampled, the structure of the Opa-CEACAM complex is needed. Towards this end, a model of the Opa60-CEACAM1 binding complex will be determined using a hybrid Double Electron Electron Resonance (DEER) and molecular dynamics (MD) approach. DEER distances between Opa60 and CEACAM1 provide spatial restraints for MD simulations of the complex, and preliminary models will be presented. Elucidating the mechanism of Opa-receptor interactions will augment our understanding of the determinants promoting the entry of a foreign body into non-phagocytic cells.

Radial Tilting of the Extracellular Domain of GLIC Revealed by EPR Spectroscopy

Signaling in the brain depends on rapid opening and closing of pentameric ligand-gated ion channels (pLGICs). Defects in these proteins are linked to a variety of diseases including myasthenia, epilepsy and sleep-disorders. In addition, these proteins are therapeutic targets for various drugs including anti-convulsants, anxiolytics, anesthetics and sedatives. While recent crystal structures of prokaryotic and eukaryotic pLGICs solved in detergent micelles have shed light on the molecular architecture of these proteins, describing their conformational dynamics in physiological lipids is essential for understanding their function. Crystal structures of GLIC and the glutamate-activated chloride channel predict that agonist-mediated channel opening is associated with a quaternary twisting and a radial un-blooming (inward tilting) of the extracellular binding domain (ECD). Using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy and functional GLIC channels reconstituted in liposomes, we are testing these gating predictions. Residues located at the top of β-strands (β1, 2, 5, 6, 8) were mutated to cysteine, labeled with the spin probe MTSL and proton-induced gating motions were measured using continuous wave (CW) and double electron-electron resonance (DEER) spectroscopy. CW EPR spectroscopy data showed the spin label attached to I92C, S93C, V94C (β5), T99C and Q101C (β6) moved into a slightly more constrained/immobile environment at pH 3.0 as compared to pH 7.6. DEER distance distributions for K48 (β2), S93 (β5), T99 and Q101 (β6) revealed the probes moved closer together at pH 3.0, suggesting that the inner β-sheet of the extracellular domain of GLIC tilt inwards upon agonist activation.

Bayesian Statistical Methods in the Analysis of DEER Data

Double Electron-Electron Resonance (DEER), also known as Pulsed Electron Double Resonance (PELDOR), is an increasingly important pulsed Electron Paramagnetic Resonance (EPR) technique for long distance (20-80 A) measurements in biomacromolecules. Notably, DEER reports a distribution of distances, P(r), corresponding to protein conformational ensembles in frozen glassy solutions. Due to the ill-conditioned, inverse nature of DEER data analysis, numerical regularization is required to obtain P(r). Further, a robust method for quantifying uncertainty in DEER distance distributions does not currently exist, complicating interpretation. Here, we compare methods of regularization parameter selection and demonstrate Bayesian statistical methods to quantify uncertainty in DEER distance distributions arising from measurement noise. We generate corresponding Bayesian credible intervals for visualization and interpretation.

Gd(3+) Spin Labels Report the Conformation and Solvent Accessibility of Solution and Vesicle-Bound Melittin.

Although Gd(3+)-based spin labels have been shown to be an alternative to nitroxides for double electron-electron resonance (DEER) distance measurements at high fields, their ability to provide solvent accessibility information, as nitroxides do, has not been explored. In addition, the effect of the label type on the measured distance distribution has not been sufficiently characterized. In this work, we extended the applicability of Gd(3+) spin labels to solvent accessibility measurements on a peptide in model membranes, namely, large unilamellar vesicles (LUVs) using W-band (2)H Mims electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) techniques and Gd(3+)-ADO3A-labeled melittin. In addition, we carried out Gd(3+)-Gd(3+) DEER distance measurements to probe the peptide conformation in solution and when bound to LUVs. A comparison with earlier results reported for the same system with nitroxide labels shows that, although in both cases the peptide binds parallel to the membrane surface, the Gd(3+)-ADO3A label tends to protrude from the membrane into the solvent, whereas the nitroxide does the opposite. This can be explained on the basis of the hydrophilicity of the Gd(3+)-ADO3A labels in contrast with the hydrophobicity of nitroxides. The distance distributions obtained from different labels are accordingly different, with the Gd(3+)-ADO3A yielding consistently broader distributions. These discrepancies are most pronounced when the peptide termini are labeled, which implies that such labeling positions may be inadvisible.

Ensemble-Biased Metadynamics: A Molecular Simulation Method to Sample Experimental Distributions

We introduce an enhanced-sampling method for molecular dynamics (MD) simulations referred to as ensemble-biased metadynamics (EBMetaD). The method biases a conventional MD simulation to sample a molecular ensemble that is consistent with one or more probability distributions known a priori, e.g., experimental intramolecular distance distributions obtained by double electron-electron resonance or other spectroscopic techniques. To this end, EBMetaD adds an adaptive biasing potential throughout the simulation that discourages sampling of configurations inconsistent with the target probability distributions. The bias introduced is the minimum necessary to fulfill the target distributions, i.e., EBMetaD satisfies the maximum-entropy principle. Unlike other methods, EBMetaD does not require multiple simulation replicas or the introduction of Lagrange multipliers, and is therefore computationally efficient and straightforward in practice. We demonstrate the performance and accuracy of the method for a model system as well as for spin-labeled T4 lysozyme in explicit water, and show how EBMetaD reproduces three double electron-electron resonance distance distributions concurrently within a few tens of nanoseconds of simulation time. EBMetaD is integrated in the open-source PLUMED plug-in (www.plumed-code.org), and can be therefore readily used with multiple MD engines.

Cysteine-specific Cu2+ chelating tags used as paramagnetic probes in double electron electron resonance.

Double electron electron resonance (DEER) is an attractive technique that is utilized for gaining insight into protein structure and dynamics via nanometer-scale distance measurements. The most commonly used paramagnetic tag in these measurements is a nitroxide spin label, R1. Here, we present the application of two types of high-affinity Cu(2+) chelating tags, based on the EDTA and cyclen metal-binding motifs as alternative X-band DEER probes, using the B1 immunoglobulin-binding domain of protein G (GB1) as a model system. Both types of tags have been incorporated into a variety of protein secondary structure environments and exhibit high spectral sensitivity. In particular, the cyclen-based tag displays distance distributions with comparable distribution widths and most probable distances within 1-3 Å when compared to homologous R1 distributions. The results display the viability of the cyclen tag as an alternative to the R1 side chain for X-band DEER distance measurements in proteins.

Combining NMR and EPR to Determine Structures of Large RNAs and Protein-RNA Complexes in Solution.

Although functional significance of large noncoding RNAs and their complexes with proteins is well recognized, structural information for this class of systems is very scarce. Their inherent flexibility causes problems in crystallographic approaches, while their typical size is beyond the limits of state-of-the-art purely NMR-based approaches. Here, we review an approach that combines high-resolution NMR restraints with lower resolution long-range constraints based on site-directed spin labeling and measurements of distance distribution restraints in the range between 15 and 80Å by the four-pulse double electron-electron resonance (DEER) EPR technique. We discuss sample preparation, the basic assumptions behind data analysis in the EPR-based distance measurements, treatment of the label-based constraints in generation of the structure, and the back-calculation of distance distributions for structure validation. Step-by-step protocols are provided for DEER distance distribution measurements including data analysis and for CYANA based structure calculation using combined NMR and EPR data.

Allosteric Regulation of the Cyclic Nucleotide-Binding Domain in HCN Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating pacemaking activity in the heart and brain. They are regulated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD). Binding of cyclic nucleotides increases the rate and extent of activation of the channels and shifts channel activation to less hyperpolarized voltages. In intact channels, cAMP and cGMP are full agonists and cCMP is a partial agonist. We use double electron-electron resonance (DEER) to study the conformational change associated with the binding of these three different cyclic nucleotide species to the CNBD. We find that conformational changes associated with ligand binding vary depending on the cyclic nucleotide bound and the location in the CNBD. We use the restrained ensemble MD (re-MD) simulations method to generate structural models integrating the complete set of experimentally measured data from DEER distance distribution histograms that describe the separation between pairs of spin labels attached to the CNBD. Our results indicate that, in the B-helix, binding of cGMP or cCMP has an effect intermediate to that of cAMP. However, in the C-helix cAMP and cCMP exhibit similar effects, but cGMP produces an intermediate effect between the apo and bound cAMP/cCMP DEER distance distributions. These data provide an interesting lens for studying allostery in HCN channels and indicate that the mechanism of protein allostery in the CNBD varies for different cyclic nucleotides.

RIDME Spectroscopy with Gd(III) Centers.

The relaxation induced dipolar modulation enhancement (RIDME) technique is applied at W-band microwave frequencies around 94 GHz to a pair of Gd(III) complexes that are connected by a rodlike spacer, and the extraction of the interspin distance distribution is discussed. A dipolar pattern derived from RIDME experimental data is a superposition of Pake-like dipolar patterns corresponding to the fundamental dipolar interaction and higher harmonics thereof. Intriguingly, the relative weights of the stretched patterns do not depend significantly on mixing time. As much larger modulation depths can be achieved than in double electron-electron resonance distance measurements at the same frequency, Gd(III)-Gd(III) RIDME may become attractive for structural characterization of biomacromolecules and biomolecular complexes.

Structural investigation of the transmembrane domain of KCNE1 in proteoliposomes.

KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel.

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.

Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies

The metallo-β-lactamases (MβLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in β-lactam-containing antibiotics, and render the antibiotics inactive. All known MβLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MβL L1 with spin labels at positions 163 and 286 and analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was “diluted” by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling.

The Role of Conformational Flexibility in Inhibitor Binding and Substrate Recognition for Cyp119

Conformational flexibility of several cytochromes P450 has been observed in response to substrate and ligand binding, and thus may play an important role in catalysis. CYP119, a bacterial thermophilic protein from cytochrome P450 superfamily has been observed in three different conformations with different inhibitors bound using X-ray crystallography, but the significance of these states in solution and in the function of the enzyme is not well known. According to the crystal structures, much of the diversity in conformational states arises in F and G helices, which fold around the substrate binding site at the distal heme face. Double electron electron resonance (DEER) was used to measure the average distances and the distance distributions between spin-labels for populated conformational states in solution. Pairs of spin-labels were introduced by coupling to engineered cysteines on the protein surface, and the effects of labeling on ligand dissociation constants (Kd) and enzyme function were characterized. DEER results from three different mutants of CYP119 indicated that 4-Phenylimidazole binding results in a conformational state that is distinct substrate-free and Imidazole-bound forms. The DEER distance changes between the two conformational states were determined as 10A for two of the mutants and 12A for the other one. Data will also be presented for the complex between CYP119 its proposed substrate, lauric acid. These results will be discussed in terms of models for substrate recognition and gated functional behavior in these enzymes.

Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy

Methods for measuring nanometer scale distances between specific sites in biomolecules (proteins and nucleic acids) and their complexes are essential for describing and analyzing their structure and function. In the last decade pulse EPR techniques were proven very effective for measuring distances between two spin labels attached to a biomolecule. The most commonly used spin labels for such measurements are nitroxide stable radicals. Recently, a new family of spin labels, based on Gd(3+) chelates, has been introduced to overcome some of the limitations of using nitroxides, particularly at high magnetic fields, which are attractive due to the increased sensitivity they offer. The benefits that such S = 7/2 spin labels offer for frequencies of 30 GHz and higher, particularly at 95 GHz, include (1) high sensitivity, only ∼0.15 nmol of doubly labeled biomolecule is needed, (2) the lack of orientation selection, which allows straightforward data analysis. Gd(3+)-Gd(3+) DEER (double electron-electron resonance) distance measurements on labeled peptides, proteins and DNA have already been demonstrated and the results show that they are very promising in terms of sensitivity. In this Perspective we review these new developments. We briefly introduce the characteristics of the DEER experiment on a pair of S = 1/2 spins and characterize the EPR spectroscopic properties of Gd(3+) ions. We then introduce some of the tags employed to attach Gd(3+) to biomolecules and provide a few experimental examples of Gd(3+)-Gd(3+) DEER measurements. This is followed by a discussion of the parameters that affect the sensitivity of such DEER measurements. Since an important term in the spin Hamiltonian of Gd(3+) is the zero-field splitting (ZFS), its effect on the DEER modulation frequencies must be considered and this is discussed next. Finally, another recently reported approach for using Gd(3+) in distance measurements will be presented: the use of Gd(3+)-nitroxide pairs.

Paramagnetic Metal Ions in Pulsed ESR Distance Distribution Measurements

The use of pulsed electron spin resonance (ESR) to measure interspin distance distributions has advanced biophysical research. The three major techniques that use pulsed ESR are relaxation rate based distance measurements, double quantum coherence (DQC), and double electron electron resonance (DEER). Among these methods, the DEER technique has become particularly popular largely because it is easy to implement on commercial instruments and because programs are available to analyze experimental data. Researchers have widely used DEER to measure the structure and conformational dynamics of molecules labeled with the methanethiosulfonate spin label (MTSSL). Recently, researchers have exploited endogenously bound paramagnetic metal ions as spin probes as a way to determine structural constraints in metalloproteins. In this context Cu(2+) has served as a useful paramagnetic metal probe at X-band for DEER based distance measurements. Sample preparation is simple, and a coordinated-Cu(2+) ion offers limited spatial flexibility, making it an attractive probe for DEER experiments. On the other hand, Cu(2+) has a broad absorption ESR spectrum at low temperature, which leads to two potential complications. First, the Cu(2+)-based DEER time domain data has lower signal to noise ratio compared with MTSSL. Second, accurate distance distribution analysis often requires high-quality experimental data at different external magnetic fields or with different frequency offsets. In this Account, we summarize characteristics of Cu(2+)-based DEER distance distribution measurements and data analysis methods. We highlight a novel application of such measurements in a protein-DNA complex to identify the metal ion binding site and to elucidate its chemical mechanism of function. We also survey the progress of research on other metal ions in high frequency DEER experiments.

Double Electron–Electron Resonance Probes Ca2+-Induced Conformational Changes and Dimerization of Recoverin

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²⁺-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²⁺-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²⁺-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²⁺-free recoverin and 3.7 ± 0.1 nm for Ca²⁺-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²⁺-bound recoverin may represent an intermolecular distance between C39 and N120. ¹⁵N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²⁺-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca²⁺-free recoverin is monomeric. We propose that Ca²⁺-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²⁺-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²⁺-induced dimerization is functionally important but not well understood.

EPR Spectroscopic Studies on the Binding of the Full Length Human KCNE1 Protein with the Voltage Gated Potassium Channel KCNQ1

KCNE1 is a single transmembrane protein that modulates the activity of voltage gated potassium channels. KCNE1 interacts with KCNQ1 and decreases the rate of channel activation, increases conductance, and generates a slowly activating K+ current critical for cardiac repolarization. Mutations on either KCNE1 or KCNQ1 genes in E1/Q1 complex can lead to cardiac long QT syndrome. Despite the biological significance of the Q1/E1 interaction, its exact nature is not fully understood yet. In this study, we have used different electron paramagnetic resonance (EPR) spectroscopic techniques to provide the direct evidence of binding activities of KCNQ1 with KCNE1. A CW-EPR titration experiment was conducted on different sites in the C-terminus of KCNQ1 with the addition of WT-KCNE1 to determine the binding mechanism of Q1 and E1. The CW EPR line shape analysis indicates that the motion of the spin label on Q1 decreases with the addition of WT-KCNE1 and saturates at molar ratio of 1:3 (Q1:E1). Double electron-electron resonance (DEER) Spectroscopy was used to measure distances between the spin labels attached on E1 and Q1 separately in Q1/E1 complex. A DEER distance of 33 A was obtained between the spin labels at 318th position on Q1 and 106th position on E1 that provides the direct evidence of formation of a Q1 and E1 complex. These EPR techniques are very useful for determining the structural model of the Q1/E1 complex.