Cu2+ based distance measurements using the double-histidine (dHis) motif by pulsed ESR present an attractive strategy to obtain precise, narrow distance distributions that can be easily related to protein backbone structure (Cunningham et al., Angew. Chem., Int. Ed., 2015, 54, 633). The Cu2+-ion is introduced as a complex with the iminodiacetic acid (IDA) chelating agent, which enhances binding selectivity to the two histidine residues that are site-selectively placed on the protein through mutagenesis. However, initial results of this method produced weak dipolar modulations. To enhance applicability of the double histidine motif using IDA, we perform a systematic examination of the possible causes of these weak dipolar modulations. We examine the efficiency of the Cu2+-ion to form the Cu2+–IDA complex in solution. In addition, we analyze the selectivity of Cu2+–IDA binding to dHis sites at both α-helical and β-strand environments. Our results indicate that the dHis motif on the β-sheet sites have high affinity towards Cu2+–IDA while the dHis sites on α-helices show poor affinity for the metal-ion complex. We are able to use our new findings to optimize conditions to maximize dHis loading while minimizing both free Cu2+ and unbound Cu2+–IDA complex in solution, allowing us to double the sensitivity of the Double Electron–Electron Resonance (DEER) experiment. Finally, we illustrate how Cu2+-based CW-ESR and DEER can be combined to obtain information on populations of different Cu2+-complexes in solution.

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 Cu2+ 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.

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 Cu2+ has served as a useful paramagnetic metal probe at X-band for DEER based distance measurements. Sample preparation is simple, and a coordinated-Cu2+ ion offers limited spatial flexibility, making it an attractive probe for DEER experiments. On the other hand, Cu2+ has a broad absorption ESR spectrum at low temperature, which leads to two potential complications. First, the Cu2+-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 Cu2+-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.

We validate the use of ESEEM to predict the number of 14N nuclei coupled to a Cu(II) ion by the use of model complexes and two small peptides with well-known Cu(II) coordination. We apply this method to gain new insight into less explored aspects of Cu(II) coordination in amyloid-β (Aβ). Aβ has two coordination modes of Cu(II) at physiological pH. A controversy has existed regarding the number of histidine residues coordinated to the Cu(II) ion in component II, which is dominant at high pH (∼8.7) values. Importantly, with an excess amount of Zn(II) ions, as is the case in brain tissues affected by Alzheimer’s disease, component II becomes the dominant coordination mode, as Zn(II) selectively substitutes component I bound to Cu(II). We confirm that component II only contains single histidine coordination, using ESEEM and set of model complexes. The ESEEM experiments carried out on systematically 15N-labeled peptides reveal that, in component II, His 13 and His 14 are more favored as equatorial ligands compared to His 6. Revealing molecular level details of subcomponents in metal ion coordination is critical in understanding the role of metal ions in Alzheimer’s disease etiology.

Modular assembly of bio-inspired supramolecular polymers is a powerful technique to develop new soft nanomaterials, and protein folding is a versatile basis for preparing such materials. Previous work demonstrated a significant difference in the physical properties of closely related supramolecular polymers composed of building blocks in which identical coiled-coil-forming peptides are cross-linked by one of two subtly different organic linkers (one flexible and the other rigid). Herein, we investigate the molecular basis for this observation by isolating a single subunit of the supramolecular polymer chain and probing its structure and conformational flexibility by double electron–electron resonance (DEER) spectroscopy. Experimental spin–spin distance distributions for two different labeling sites coupled with molecular dynamics simulations provide insights into how the linker structure impacts chain dynamics in the coiled-coil supramolecular polymer.

The interaction of Cu(II) and Zn(II) ions with amyloid-β (Aβ) plays an important role in the etiology of Alzheimer’s disease. We describe the use of electron spin resonance (ESR) to measure metal-binding competition between Cu(II) and Zn(II) in amyloid-β at physiological pH. Continuous wave ESR measurements show that the affinity of Cu(II) toward Aβ(1–16) is significantly higher than that of Zn(II) at physiological pH. Importantly, of the two known Cu(II) coordination modes in Aβ, component I and component II, Zn(II) displaces Cu(II) only from component I. Our results indicate that at excess amounts of Zn(II) component II becomes the most dominant coordination mode. This observation is important as Aβ aggregates in the brain contain a high Zn(II) ion concentration. In order to determine details of the metal ion competition, electron spin echo envelope modulation experiments were carried out on Aβ variants that were systematically 15N labeled. In the presence of Zn(II), most peptides use His 14 as an equatorial ligand to bind Cu(II) ions. Interestingly, Zn(II) ions completely substitute Cu(II) ions that are simultaneously coordinated to His 6 and His 13. Furthermore, in the presence of Zn(II), the proportion of Cu(II) ions that are simultaneously coordinated to His 13 and His 14 is increased. On the basis of our results we suggest that His 13 plays a critical role in modulating the morphology of Aβ aggregates.

Double quantum coherence (DQC) ESR spectroscopy is applied to measure the Cu2+–Cu2+ distance in the EcoRI-DNA complex. A simple method is proposed to reduce the contribution of nuclear hyperfine and quadrupole interactions to such data. The effects of such interactions between the electron spin of Cu2+ and neighboring nuclei on the DQC data make it difficult to measure the nanometer range interspin distance. The DQC data is in good agreement with results obtained by double electron electron resonance (DEER) spectroscopy. At the same time, the signal-to-noise ratio per shot in DQC is high. Taken together, these results provide impetus for further development of paramagnetic metal ion-based DQC techniques.

X-ray crystallography has been a useful tool in the development of site-directed spin labeling by resolving rotamers of the nitroxide spin-label side chain in a variety of α-helical environments. In this work, the crystal structure of a doubly spin-labeled N8C/K28C mutant of the B1 immunoglobulin-binding domain of protein G (GB1) was solved. The double mutant formed a domain-swapped dimer under crystallization conditions. Two rotameric states of the spin-label were resolved at the solvent-exposed α-helical site, at residue 28; these are in good agreement with rotamers previously reported for helical structures. The second site, at residue 8 on an interior β-strand, shows the presence of three distinct solvent-exposed side-chain rotamers. One of these rotamers is rarely observed within crystal structures of R1 sites and suggests that the Hα and Sδ hydrogen bond that is common to α-helical sites is absent at this interior β-strand residue. Variable temperature continuous wave (CW) experiments of the β-strand site showed two distinct components that were correlated to the rotameric states observed in crystallography. Interestingly, the CW data at room temperature could be fit without the use of an order parameter, which is consistent with the lack of the Hα and Sδ interaction. Additionally, double electron electron resonance (DEER) spectroscopy was performed on the GB1 double mutant in its monomeric form and yielded a most probable interspin distance of 25 ± 1 Å. In order to evaluate the accuracy of the measured DEER distance, the rotamers observed in the crystal structure of the domain-swapped GB1 dimer were modeled into a high-resolution structure of the wild type monomeric GB1. The distances generated in the resulting GB1 structural models match the most probable DEER distance within ∼2 Å. The results are interesting as they indicate by direct experimental measurement that the rotameric states of R1 found in this crystal provide a very close match to the most probable distance measured by DEER.

Site-directed spin labeling, wherein a nitroxide side chain is introduced into a protein at a selected mutant site, is increasingly employed to investigate biological systems by electron spin resonance (ESR) spectroscopy. An understanding of the packing and dynamics of the spin label is needed to extract the biologically relevant information about the macromolecule from ESR measurements. In this work, molecular dynamics (MD) simulations were performed on the spin-labeled restriction endonuclease, EcoRI in complex with DNA. Mutants of this homodimeric enzyme were previously constructed, and distance measurements were performed using the double electron electron resonance experiment. These correlated distance constraints have been leveraged with MD simulations to learn about side chain packing and preferred conformers of the spin label on sites in an α-helix and a β-strand. We found three dihedral angles of the spin label side chain to be most sensitive to the secondary structure where the spin label was located. Conformers sampled by the spin label differed between secondary structures as well. Cα–Cα distance distributions were constructed and used to extract details about the protein backbone mobility at the two spin labeled sites. These simulation studies enhance our understanding of the behavior of spin labels in proteins and thus expand the ability of ESR spectroscopy to contribute to knowledge of protein structure and dynamics.

The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu2+ ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu2+ coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu2+-ion based long-range distance constraints. Double electron-electron resonance data yield Cu2+-Cu2+ and Cu2+-nitroxide distances that are uniquely consistent with one Cu2+ bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu2+ ions bind per complex. Unexpectedly, Mg2+-catalyzed DNA cleavage by EcoRI is profoundly inhibited by Cu2+ binding at these hitherto unknown sites, 13 Å away from the Mg2+ positions in the catalytic centers. Molecular dynamics simulations suggest a model for inhibition of catalysis, whereby the Cu2+ ions alter critical protein-DNA interactions and water molecule positions in the catalytic sites. In the absence of Cu2+, the Mg2+-dependence of EcoRI catalysis shows positive cooperativity, which would enhance EcoRI inactivation of foreign DNA by irreparable double-strand cuts, in preference to readily repaired single-strand nicks. Nonlinear Poisson-Boltzmann calculations suggest that this cooperativity arises because the binding of Mg2+ in one catalytic site makes the surface electrostatic potential in the distal catalytic site more negative, thus enhancing binding of the second Mg2+. Taken together, our results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.

Tau protein and Cu(II) are believed to be associated with the pathogenesis of Alzheimer’s disease. However, little is known about atomic-level interactions between tau protein and Cu(II). Herein, we suggest, on the basis of electron spin resonance (ESR) data, that the four pseudorepeats of tau protein in the microtubule-binding region play an important role in Cu(II) binding. We use a number of tau protein fragments in order to examine Cu(II)-binding site(s) and binding affinities. Continuous-wave (CW) ESR experiments on the four highly conserved octadecapeptides, each of which is a segment of one of the four pseudorepeats, reveal that the equimolar Cu(II) complexes of the four octadecapeptides are similar to one another in terms of the coordination environment and binding affinity. The spectra obtained with pulsed ESR techniques such as electron spin–echo envelope modulation and hyperfine sublevel correlation provide direct evidence that a histidine residue and a backbone amide group coordinate to Cu(II) in each Cu(II)–octadecapeptide complex. The results of CW and pulsed ESR experiments on some chemically modified peptides indicate that the cysteine residues in the second and third pseudorepeats are unlikely to be involved in Cu(II) binding. On the other hand, similar experiments on tau fragments of the second pseudorepeat with different lengths lead to the conclusion that the affinity for Cu(II) decreases as the octadecapeptide is either truncated or elongated. The high Cu(II)-binding affinity of the octadecapeptide is presumably due to the N-terminal amino group stabilizing the Cu(II)–octadecapeptide complex. Finally, the ESR data for a longer tau fragment that contains two octadecapeptides suggest that the Cu(II) binding site(s) of even longer fragments of tau protein is similar to that of a single octadecapeptide.

The interaction of amyloid-β (Aβ) peptide with Cu(II) appears to play an important role in the etiology of Alzheimer’s disease. At physiological pH, the Cu(II) coordination in Aβ is heterogeneous, and there exist at least two binding modes in which Cu(II) is coordinated by histidine residues. Electron spin resonance studies have revealed a picture of the Cu(II) binding at a higher or lower pH, where only one of the two binding modes is almost exclusively present. We describe a procedure to directly examine the coordination of Cu(II) to each histidine residue in the dominant binding mode at physiological pH. We use nonlabeled and residue-specifically 15N-labeled Aβ(1−16). For quantitative analysis, the intensities of three-pulse electron spin-echo envelope modulation (ESEEM) spectra are analyzed. Spectral simulations show that ESEEM intensities provide information about the contribution of each histidine residue. Indeed, the ESEEM experiments at pH 6.0 confirm the dominant contribution of His6 to the Cu(II) coordination as expected from the work of other researchers. Interestingly, however, the ESEEM data obtained at pH 7.4 reveal that the contributions of the three residues to the Cu(II) coordination are in the order of His14 ≈ His6 > His13 in the dominant binding mode. The order indicates a significant contribution from the simultaneous coordination by His13 and His14 at physiological pH, which has been underappreciated. These findings are supported by hyperfine sublevel correlation spectroscopy experiments. The simultaneous coordination by the two adjacent residues is likely to be present in a non-β-sheet structure. The coexistence of different secondary structures is possibly the molecular origin for the formation of amorphous aggregates rather than fibrils at relatively high concentrations of Cu(II). Through our approach, precise and useful information about Cu(II) binding in Aβ(1−16) at physiological pH is obtained without any side-chain modification, amino acid residue replacement, or pH change, each of which might lead to an alteration in the peptide structure or the coordination environment.

We present the measurement of Cu2+−Cu2+ and Cu2+−nitroxide distance distributions using double electron−electron resonance (DEER) on a proline-based peptide and an alanine-based peptide. The proline-based peptide contains two well-characterized Cu2+ binding segments, PHGGGW, separated by seven proline residues. The alanine-based peptide contains a PHGGGW segment at one end of the peptide and a nitroxide spin label attached to a cysteine residue close to the other end of the peptide. DEER experiments were performed at several external magnetic fields and resonance offsets to probe the orientational effects on the Cu2+-based DEER signal. Subtle but detectable orientational effects were observed from the DEER spectra of both peptides. A general theoretical model was developed to analyze the experimental data sets. We show that the Tikhonov regularization-based method is not applicable to extract precise Cu2+-based distance distributions. Instead, a full data analysis is required to obtain the distance distributions and relative orientations between spin centers. A 30 Å mean Cu2+−Cu2+ distance and a 27 Å mean Cu2+−nitroxide distance were determined in the two peptides. These distances are consistent with structural models and with earlier measurements. Constraints on the relative orientation between paramagnetic centers in these two model peptides were determined by examination of the orientational effects. The data analysis procedure is system independent, and therefore is applicable to more complicated biological systems.

The overall morphology and Cu(II) ion coordination for the aggregated amyloid-β(1−40) [Aβ(1−40)] in N-ethylmorpholine (NEM) buffer are affected by Cu(II) ion concentration. This effect is investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and electron spin echo envelope modulation (ESEEM) spectroscopy. At lower than equimolar concentrations of Cu(II) ions, fibrillar aggregates of Aβ(1−40) are observed. At these concentrations of Cu(II), the monomeric and fibrillar Aβ(1−40) ESEEM data indicate that the Cu(II) ion is coordinated by histidine residues. For aggregated Aβ(1−40) at a Cu(II):Aβ molar ratio of 2:1, TEM and AFM images show both linear fibrils and granular amorphous aggregates. The ESEEM spectra show that the multi-histidine coordination for Cu(II) ion partially breaks up and becomes exposed to water or exchangeable protons of the peptide at a higher Cu(II) concentration. Since the continuous-wave electron spin resonance results also suggest two copper-binding sites in Aβ(1−40), the proton ESEEM peak may arise from the second copper-binding site, which may be significantly involved in the formation of granular amorphous aggregates. Thioflavin T fluorescence and circular dichroism experiments also show that Cu(II) inhibits the formation of fibrils and induces a nonfibrillar β-sheet conformation. Therefore, we propose that Aβ(1−40) has a second copper-binding site in a proton-rich environment and the second binding Cu(II) ion interferes with a conformational transition into amyloid fibrils, inducing the formation of granular amorphous aggregates.

We provide direct evidence that all three histidine residues in amyloid-β1−16 (Aβ1−16) coordinate to Cu(II). In our approach, we generate Aβ1−16 analogues, in each of which a selected histidine residue is isotopically enriched with 15N. Pulsed electron spin resonance (ESR) experiments such as electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectroscopy clearly show that all three histidine imidazole rings at positions 6, 13 and 14 in Aβ1−16 bind to Cu(II). The method employed here does not require either chemical side chain modification or amino acid residue replacement, each of which is traditionally used to determine whether an amino acid residue in a protein binds to a metal ion. We find that the histidine coordination in the Aβ1−16 peptide is independent of the Cu(II)-to-peptide ratio, which is in contrast to the Aβ1−40 peptide. The ESR results also suggest tight binding between the histidine residues and the Cu(II) ion, which is likely the reason for the high binding affinity of the Aβ peptide for Cu(II).

We demonstrate the synthesis of a series of spin-labeled curved oligomers to determine their end-to-end lengths and distance distributions using electron spin resonance. We synthesize shape-persistent macromolecules from conformationally restricted, asymmetric monomers that are coupled through pairs of amide bonds to create water-soluble, spiro-ladder oligomers with well-defined three-dimensional structures. We synthesized seven different macromolecules, each containing eight monomers but differing in the sequence to create macromolecules with different curved shapes. The ends of the oligomers were labeled with nitroxide spin probes, and double electron−electron resonance (DEER) electron spin resonance (ESR) experiments were carried out to obtain quantitative information about the shapes and flexibility of the oligomers. The most probable end-to-end distance of the oligomers ranges from 23 to 36 Å, a range of length that we previously accessed by assembling rod-like homo-oligomers that contain 4−8 bisamino acid monomers. The relative distances measured for the oligomers confirm that, by varying the sequence of an oligomer, we are able to control its shape. The shapes of the ESR-derived population distributions allow us to compare the degree of shape persistence and flexibility of spiro-ladder oligomers to other well-studied nanoscale molecular structures such as p-phenylethynylenes.Keywords: bispeptides; curved nanostructures; EPR spectroscopy; molecular flexibility; nanostructures; organic nanostructures; spin probes

Herein, we identify the coordination environment of Cu2+ in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn2+ and Cu2+ significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn2+ can either potentiate or inhibit GlyR activity depending on its concentration, while Cu2+ is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu2+ coordination and stoichiometry. We show that Cu2+ has one binding site per α1 subunit, and that five Cu2+ can be coordinated per GlyR. Cu2+ binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu2+ coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn2+ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.