Sunil Saxena

Amplification of xenon NMR and MRI by remote detection

A technique is proposed in which an NMR spectrum or MRI is encoded and stored as spin polarization and is then moved to a different physical location to be detected. Remote detection allows the separate optimization of the encoding and detection steps, permitting the independent choice of experimental conditions and excitation and detection methodologies. In the initial experimental demonstration of this technique, we show that taking dilute 129Xe from a porous sample placed inside a large encoding coil and concentrating it into a smaller detection coil can amplify NMR signal. In general, the study of NMR active molecules at low concentration that have low physical filling factor is facilitated by remote detection. In the second experimental demonstration, MRI information encoded in a very low-field magnet (4–7 mT) is transferred to a high-field magnet (4.2 T) to be detected under optimized conditions. Furthermore, remote detection allows the utilization of ultrasensitive optical or superconducting quantum interference device detection techniques, which broadens the horizon of NMR experimentation.

Double quantum two-dimensional Fourier transform electron spin resonance: Distance measurements

Abstract The first double quantum two-dimensional Fourier transform electron spin resonance (2D-FT ESR) experiments are reported. Extension of 2D-FT ESR to enable detection of the ΔM s = ±2transition enhances the capability of ESR in studying structural properties (i.e. distances in bilabeled molecules). A distance of 21 A was found for a poly-proline peptide, spin labeled at both ends, in agreement with earlier measurements using fluorescence energy transfer.

Theory of double quantum two-dimensional electron spin resonance with application to distance measurements

A formulation is presented for calculating double quantum two dimensional electron spin resonance (DQ-2D ESR) spectra in the rigid limit that correspond to recent experimental DQ-2D ESR spectra obtained from a nitroxide biradical. The theory includes the dipolar interaction between the nitroxide moieties as well as the fully asymmetric g and hyperfine tensors and the angular geometry of the biradical. The effects of arbitrary pulses (strong but not truly nonselective pulses) are included by adapting the recently introduced split Hamiltonian theory for numerical simulations. It is shown how arbitrary pulses in magnetic resonance create “forbidden” coherence pathways, and their role in DQ-2D ESR is delineated. The high sensitivity of these DQ-2D ESR signals to the strength of the dipolar interaction is demonstrated and rationalized in terms of the orientational selectivity of the “forbidden” pathways. It is further shown that this selectivity also provides constraints on the structural geometry (i.e., the orientations of the nitroxide moieties) of the biradicals. The theory is applied to the recent double quantum modulation (DQM) experiment on an end-labeled poly-proline peptide biradical. A distance of 18.5 A between the ends is found for this biradical. A new two pulse double quantum experiment is proposed (by analogy to recent NMR experiments), and its feasibility for the ESR case is theoretically explored.

Direct Evidence That All Three Histidine Residues Coordinate to Cu(II) in Amyloid-β1−16†

We provide direct evidence that all three histidine residues in amyloid-beta 1-16 (Abeta 1-16) coordinate to Cu(II). In our approach, we generate Abeta 1-16 analogues, in each of which a selected histidine residue is isotopically enriched with (15)N. 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 Abeta 1-16 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 Abeta 1-16 peptide is independent of the Cu(II)-to-peptide ratio, which is in contrast to the Abeta 1-40 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 Abeta peptide for Cu(II).

ESR spectroscopy identifies inhibitory Cu2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

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.

Substantial Contribution of the Two Imidazole Rings of the His13−His14 Dyad to Cu(II) Binding in Amyloid-β(1−16) at Physiological pH and Its Significance

The interaction of amyloid-β (Aβ) peptide with Cu(II) appears to play an important role in the etiology of Alzheimers 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 (15)N-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.

An approach towards the measurement of nanometer range distances based on Cu2+ ions and ESR.

We present the measurement of Cu(2+)-Cu(2+) and Cu(2+)-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 Cu(2+) 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 Cu(2+)-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 Cu(2+)-based distance distributions. Instead, a full data analysis is required to obtain the distance distributions and relative orientations between spin centers. A 30 A mean Cu(2+)-Cu(2+) distance and a 27 A mean Cu(2+)-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 Second Cu(II)-Binding Site in a Proton-Rich Environment Interferes with the Aggregation of Amyloid-β(1―40) into Amyloid Fibrils

The overall morphology and Cu(II) ion coordination for the aggregated amyloid-beta(1-40) [Abeta(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 Abeta(1-40) are observed. At these concentrations of Cu(II), the monomeric and fibrillar Abeta(1-40) ESEEM data indicate that the Cu(II) ion is coordinated by histidine residues. For aggregated Abeta(1-40) at a Cu(II):Abeta 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 Abeta(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 beta-sheet conformation. Therefore, we propose that Abeta(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.

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.

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

The development of ESR methods that measure long-range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein-backbone structure. Herein we present the double-histidine (dHis) Cu(2+)-binding motif as a rigid spin probe for double electron-electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X-ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu(2+) DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein-backbone structure and flexibility.