Design of the elusive proteinaceous oxygen donor copper site suggests a promising future for copper for MRI contrast agents.

We report the preparation and spectroscopic characterization of a highly elusive copper site bound exclusively to oxygen donor atoms within a protein scaffold. Despite copper generally being considered unsuitable for use in MRI contrast agents, which in the clinic are largely Gd(III) based, the designed copper coiled coil displays relaxivity values equal to, or superior than, those of the Gd(III) analog at clinical field strengths. The creation of this new-to-biology proteinaceous CuOx-binding site demonstrates the power of the de novo peptide design approach to access chemistry for abiological applications, such as for the development of MRI contrast agents.

A Gadolinium Spin Label with Both a Narrow Central Transition and Short Tether for Use in Double Electron Electron Resonance Distance Measurements.

The design, synthesis, and application of a nine-coordinate gadolinium(III)-containing spin label, [Gd.sTPATCN]-SL, for use in nanometer-distance measurement experiments by EPR spectroscopy is presented. The spin label links to cysteines via a short thioether tether and has a narrow central transition indicative of small zero-field splitting (ZFS). A protein homodimer, TRIM25cc, was selectively labeled with [Gd.sTPATCN]-SL (70%) and a nitroxide (30%) under mild conditions and measured using the double electron electron resonance (DEER) technique with both commercial Q-band and home-built W-band spectrometers. The label shows great promise for increasing the sensitivity of DEER measurements through both its favorable relaxation parameters and the large DEER modulation depth at both Q- and W-band for the inter-Gd(III) DEER measurement which, at 9%, is the largest recorded under these conditions.

Orientation selection in high-field RIDME and PELDOR experiments involving low-spin CoII ions.

Orientation selective (OS) RIDME and PELDOR were conducted on a low-spin CoII complex coordinated by two nitroxide (NO) labelled 2,2':6',2''-terpyridine ligands. Co-NO RIDME at W- and Q-band gave insight into the relative orientation between the Co-NO interspin vector (rCo-NO) and the NO moiety. This was further supported by W-band Co-NO PELDOR that also allowed elucidating the relative orientation of the CoII and NO g-tensors. Differences to earlier predictions were confirmed by DFT calculations. Finally, NO-NO PELDOR allowed retrieving the mutual orientations between the NO-NO interspin vector (rNO-NO) and the NO moieties. The results demonstrate that OS-RIDME and -PELDOR can provide geometric structure information on a system containing a CoII ion and two nitroxides. Especially, the high sensitivity and ease of interpretation of RIDME at W-band opens avenues for new applications of CoII as orthogonal spin label.

Large cross-effect dynamic nuclear polarisation enhancements with kilowatt inverting chirped pulses at 94 GHz.

Dynamic nuclear polarisation (DNP) is a process that transfers electron spin polarisation to nuclei by applying resonant microwave radiation, and has been widely used to improve the sensitivity of nuclear magnetic resonance (NMR). Here we demonstrate new levels of performance for static cross-effect proton DNP using high peak power chirped inversion pulses at 94 GHz to create a strong polarisation gradient across the inhomogeneously broadened line of the mono-radical 4-amino TEMPO. Enhancements of up to 340 are achieved at an average power of a few hundred mW, with fast build-up times (3 s). Experiments are performed using a home-built wideband kW pulsed electron paramagnetic resonance (EPR) spectrometer operating at 94 GHz, integrated with an NMR detection system. Simultaneous DNP and EPR characterisation of other mono-radicals and biradicals, as a function of temperature, leads to additional insights into limiting relaxation mechanisms and give further motivation for the development of wideband pulsed amplifiers for DNP at higher frequencies.

Navigating the perfect storm: research strategies for socialecological systems in a rapidly evolving world.

The 'Perfect Storm' metaphor describes a combination of events that causes a surprising or dramatic impact. It lends an evolutionary perspective to how social-ecological interactions change. Thus, we argue that an improved understanding of how social-ecological systems have evolved up to the present is necessary for the modelling, understanding and anticipation of current and future social-ecological systems. Here we consider the implications of an evolutionary perspective for designing research approaches. One desirable approach is the creation of multi-decadal records produced by integrating palaeoenvironmental, instrument and documentary sources at multiple spatial scales. We also consider the potential for improved analytical and modelling approaches by developing system dynamical, cellular and agent-based models, observing complex behaviour in social-ecological systems against which to test systems dynamical theory, and drawing better lessons from history. Alongside these is the need to find more appropriate ways to communicate complex systems, risk and uncertainty to the public and to policy-makers.

High power pulsed dynamic nuclear polarisation at 94 GHz.

In this communication we report initial results using high power pulsed techniques at 94 GHz to perform solid state Dynamic Nuclear Polarisation (DNP) on high volume samples. It is shown that excitation with short pulses, comparable to the pi/2 pulse length, at fast repetition rates can result in higher DNP enhancements relative to continuous wave (cw) excitation for the same average power. Peak enhancements are observed at an average power of only a few hundred mW delivered to the sample.

High-sensitivity Gd3+-Gd3+ EPR distance measurements that eliminate artefacts seen at short distances.

Gadolinium complexes are attracting increasing attention as spin labels for EPR dipolar distance measurements in biomolecules and particularly for in-cell measurements. It has been shown that flip-flop transitions within the central transition of the high-spin Gd3+ ion can introduce artefacts in dipolar distance measurements, particularly when measuring distances less than 3 nm. Previous work has shown some reduction of these artefacts through increasing the frequency separation between the two frequencies required for the double electron-electron resonance (DEER) experiment. Here we use a high-power (1 kW), wideband, non-resonant system operating at 94 GHz to evaluate DEER measurement protocols using two stiff Gd(III) rulers, consisting of two bis-Gd3+-PyMTA complexes, with separations of 2.1 nm and 6.0 nm, respectively. We show that by avoiding the -12→12 central transition completely, and placing both the pump and the observer pulses on either side of the central transition, we can now observe apparently artefact-free spectra and narrow distance distributions, even for a Gd-Gd distance of 2.1 nm. Importantly we still maintain excellent signal-to-noise ratio and relatively high modulation depths. These results have implications for in-cell EPR measurements at naturally occurring biomolecule concentrations.

EPR spectroscopy of a family of Cr(III) 7M(II) (M = Cd, Zn, Mn, Ni) "wheels": studies of isostructural compounds with different spin ground states.

Spinning wheels: The presented highly resolved multifrequency continuous wave EPR spectra (e.g., see figure) of the heterooctametalic "wheels" Cr(7)M provide rare examples of high nuclearity polymetallic systems where detailed information on the spin-Hamiltonian parameters of the ground and excited spin states is observed.We present highly resolved multifrequency (X-, K-, Q- and W-band) continous wave EPR spectra of the heterooctametalic "wheels", [(CH(3))(2)NH(2)][Cr(III) (7)M(II)F(8)((CH(3))(3)CCOO)(16)], hereafter Cr(7)M, where M=Cd, Zn, Mn, and Ni. These experimental spectra provide rare examples of high nuclearity polymetallic systems where detailed information on the spin-Hamiltonian parameters of the ground and excited spin states is observed. We interpret the EPR spectra by use of restricted size effective subspaces obtained by the rigorous solution of spin-Hamiltonians of dimension up to 10(5) by use of the Davidson algorithm. We show that transferability of spin-Hamiltonian parameters across complexes of the Cr(7)M family is possible and that the spin-Hamiltonian parameters of Cr(7)M do not have sharply defined values, but are rather distributed around a mean value.

Force detected electron spin resonance at 94 GHz.

Force detected electron spin resonance (FDESR) detects the presence of unpaired electrons in a sample by measuring the change in force on a mechanical resonator as the magnetization of the sample is modulated under magnetic resonance conditions. The magnetization is coupled to the resonator via a magnetic field gradient. It has been used to both detect and image distributions of electron spins, and it offers both extremely high absolute sensitivity and high spatial imaging resolution. However, compared to conventional induction mode ESR the technique also has a comparatively poor concentration sensitivity and it introduces complications in interpreting and combining both spectroscopy and imaging. One method to improve both sensitivity and spectral resolution is to operate in high magnetic fields in order to increase the sample magnetization and g-factor resolution. In this article we present FDESR measurements on the organic conductor (fluoranthene)(2)PF(6) at 3.2 T, with a corresponding millimeter-wave frequency of 93.5 GHz, which we believe are the highest field results for FDESR reported in the literature to date. A magnet-on-cantilever approach was used, with a high-anisotropy microwave ferrite as the gradient source and employing cyclic saturation to modulate the magnetization at the cantilever fundamental frequency.

The use of composite pulses for improving DEER signal at 94GHz.

The sensitivity of pulsed electron paramagnetic resonance (EPR) measurements on broad-line paramagnetic centers is often limited by the available excitation bandwidth. One way to increase excitation bandwidth is through the use of chirp or composite pulses. However, performance can be limited by cavity or detection bandwidth, which in commercial systems is typically 100-200MHz. Here we demonstrate in a 94GHz spectrometer, with >800MHz system bandwidth, an increase in signal and modulation depth in a 4-pulse DEER experiment through use of composite rather than rectangular π pulses. We show that this leads to an increase in sensitivity by a factor of 3, in line with theoretical predictions, although gains are more limited in nitroxide-nitroxide DEER measurements.

Atomic-molecular superlattices.

In this communication we demonstrate a directly-bonded crystalline fullerene superlattice and show that the incorporation of spin-active N@C60 endohedral fullerenes is readily achieved to give an atomic-molecular hybrid spin-active superlattice material.

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields.

High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(∼94 GHz) and D-band (∼140 GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100 ps to 2 ns regime. However, low concentration sensitivity remains a roadblock for studying aqueous samples at high magnetic fields. Here, we examine the sensitivity of a non-resonant thin-layer cylindrical sample holder, coupled to a quasi-optical induction-mode W-band EPR spectrometer (HiPER), for continuous wave (CW) EPR analyses of: (i) the aqueous nitroxide standard, TEMPO; (ii) the unstructured to α-helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232 nt RNA. For sample volumes of ∼50 µL, concentration sensitivities of 2-20 µM were achieved, representing a ∼10-fold enhancement compared to a cylindrical TE011 resonator on a commercial Bruker W-band spectrometer. These results therefore highlight the sensitivity of the thin-layer sample holders employed in HiPER for spin-labeling studies of biological macromolecules at high fields, where applications can extend to other systems that are facilitated by the modest sample volumes and ease of sample loading and geometry.

DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR.

This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron-electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.

W-band PELDOR with 1 kW microwave power: molecular geometry, flexibility and exchange coupling.

A technique that is increasingly being used to determine the structure and conformational flexibility of biomacromolecules is Pulsed Electron-Electron Double Resonance (PELDOR or DEER), an Electron Paramagnetic Resonance (EPR) based technique. At X-band frequencies (9.5 GHz), PELDOR is capable of precisely measuring distances in the range of 1.5-8 nm between paramagnetic centres but the orientation selectivity is weak. In contrast, working at higher frequencies increases the orientation selection but usually at the expense of decreased microwave power and PELDOR modulation depth. Here it is shown that a home-built high-power pulsed W-band EPR spectrometer (HiPER) with a large instantaneous bandwidth enables one to achieve PELDOR data with a high degree of orientation selectivity and large modulation depths. We demonstrate a measurement methodology that gives a set of PELDOR time traces that yield highly constrained data sets. Simulating the resulting time traces provides a deeper insight into the conformational flexibility and exchange coupling of three bisnitroxide model systems. These measurements provide strong evidence that W-band PELDOR may prove to be an accurate and quantitative tool in assessing the relative orientations of nitroxide spin labels and to correlate those orientations to the underlying biological structure and dynamics.

A spectrometer designed for 6.7 and 14.1 T DNP-enhanced solid-state MAS NMR using quasi-optical microwave transmission.

A Dynamic Nuclear Polarisation (DNP) enhanced solid-state Magic Angle Spinning (MAS) NMR spectrometer operating at 6.7 T is described and demonstrated. The 187 GHz TE(13) fundamental mode of the FU CW VII gyrotron is used as the microwave source for this magnetic field strength and 284 MHz (1)H DNP-NMR. The spectrometer is designed for use with microwave frequencies up to 395 GHz (the TE(16) second-harmonic mode of the gyrotron) for DNP at 14.1T (600 MHz (1)H NMR). The pulsed microwave output from the gyrotron is converted to a quasi-optical Gaussian beam using a Vlasov antenna and transmitted to the NMR probe via an optical bench, with beam splitters for monitoring and adjusting the microwave power, a ferrite rotator to isolate the gyrotron from the reflected power and a Martin-Puplett interferometer for adjusting the polarisation. The Gaussian beam is reflected by curved mirrors inside the DNP-MAS-NMR probe to be incident at the sample along the MAS rotation axis. The beam is focussed to a ~1 mm waist at the top of the rotor and then gradually diverges to give much more efficient coupling throughout the sample than designs using direct waveguide irradiation. The probe can be used in triple channel HXY mode for 600 MHz (1)H and double channel HX mode for 284 MHz (1)H, with MAS sample temperatures ≥85 K. Initial data at 6.7 T and ~1 W pulsed microwave power are presented with (13)C enhancements of 60 for a frozen urea solution ((1)H-(13)C CP), 16 for bacteriorhodopsin in purple membrane ((1)H-(13)C CP) and 22 for (15)N in a frozen glycine solution ((1)H-(15)N CP) being obtained. In comparison with designs which irradiate perpendicular to the rotation axis the approach used here provides a highly efficient use of the incident microwave beam and an NMR-optimised coil design.

A kilowatt pulsed 94 GHz electron paramagnetic resonance spectrometer with high concentration sensitivity, high instantaneous bandwidth, and low dead time.

We describe a quasioptical 94 GHz kW pulsed electron paramagnetic resonance spectrometer featuring pi/2 pulses as short as 5 ns and an instantaneous bandwidth of 1 GHz in nonresonant sample holders operating in induction mode and at low temperatures. Low power pulses can be as short as 200 ps and kilowatt pulses as short as 1.5 ns with timing resolution of a few hundred picoseconds. Phase and frequency can be changed on nanosecond time scales and complex high power pulse sequences can be run at repetition rates up to 80 kHz with low dead time. We demonstrate that the combination of high power pulses at high frequencies and nonresonant cavities can offer excellent concentration sensitivity for orientation selective pulsed electron double resonance (double electron-electron resonance), where we demonstrate measurements at 1 microM concentration levels.

Magnetic and high-frequency EPR studies of an octahedral Fe(III) compound with unusual zero-field splitting parameters.

Temperature-dependent magnetic susceptibility and multi-frequency EPR (9.4, 34.5, 94 and 188 GHz) spectroscopic measurements have been carried out together with an X-ray study at 100 K to study [Fe(DMSO)(6)](NO(3))(3). The iron(III) ion remains high-spin (S = 5/2) in the temperature range studied, therefore, the EPR data were interpreted using the conventional S = 5/2 spin Hamiltonian. A full analysis of EPR spectra at 95 GHz of a powdered sample at 290 K revealed that they are extremely sensitive to D and E values. The zfs parameters were precisely determined: D = + 0.1730 cm(-1), E = 0.00 cm(-1) and lambda = |E/D| = 0.00. A sequence of the spectra neatly shows that the compound has a clear magnetic dependence on temperature. The study at 5 K, showed that the zfs parameters increase: D = + 0.1970 cm(-1), E = 0.017 cm(-1) and lambda = |E/D| = 0.086. These data indicate that as the temperature decreases the D tensor increases slightly showing an increase in the rhombicity. These results confirm that |2D| is congruent to h nu at X-band in this case. Additionally, it has been shown by X-ray crystal analysis of [Fe(DMSO)(6)](NO(3))(3) at 100 K that this is involved in a hydrogen bonding network, consisting of C-H...O interactions between the nitrate anions and the methyl groups of the coordinated DMSO molecules, thus suggesting that the differences found in the spectroscopic parameters D and E at different temperatures must be due to these supramolecular interactions.

High-field EPR study and crystal and molecular structure of trans-RSSR-[CrCl2(cyclam)]nX (X=ZnCl4 2-, Cl- and Cl-.4H2O.0.5HCl).

For the first time, HF-EPR (94.5 GHz) spectroscopy has been used to determine crystal field parameters in chromium(III) coordination compounds. The large zero-field splitting parameters of the dark-green photochromic trans-RSSR-[CrCl(2)(cyclam)](2)ZnCl(4), 1, the red-purple trans-RSSR-[CrCl(2)(cyclam)]Cl, 2, and the red-purple trans-RSSR-[CrCl(2)(cyclam)]Cl.4H(2)O.0.5HCl, 3, where cyclam = 1,4,8,11-tetraazacyclotetradecane, have been obtained. A full analysis of EPR spectra at 94.5 GHz of diluted complexes 1, 2 and 3 at 300 K revealed that they are extremely sensitive to D and E values. The rhombic distortion was precisely determined for each compound. For 1, g= 2.01, D=-0.305 cm(-1), E= 0.041 cm(-1) and lambda=|E/D|= 0.1396; for 2, g= 2.01; D=-0.348 cm(-1), E= 0.042 cm(-1) and lambda=|E/D|= 0.1206 and for 3, g= 1.99, D=-0.320 cm(-1), E= 0.041 cm(-1) and ambda=|E/D|= 0.1281. The EPR study at 94.5 GHz at 10 K allowed us to confirm the sign of the D value for all compounds. These data indicate that at room temperature the crystal field is mainly rhombic and as the temperature decreases, the rhombicity of the D tensor increases slightly. These found differences between 1, 2 and 3 allowed us to establish the importance of the intermolecular interactions in the solid state due to different hydrogen bonding networks in their crystalline arrangement.

Magnetic and optical studies on an S = 6 ground-state cluster [Cr12O9(OH)3(O2CCMe3)15]: determination of, and the relationship between, single-ion and cluster spin Hamiltonian parameters.

The dodecametallic Cr(III) cluster [Cr(12)O(9)(OH)(3)(O(2)CCMe(3))(15)] has a ground spin state of S = 6 characterized by the spin Hamiltonian parameters g(ZZ)() = 1.965, g(XX)() = g(YY)() = 1.960, D(S=)()(6) = +0.088 cm(-)(1), and E(S=)()(6) = 0 (where D and E are the axial and rhombic zero-field splitting parameters, respectively) as determined by multifrequency EPR spectroscopy and magnetization studies. Micro-SQUID magnetization studies reveal steps due to the fine structure of the ground state, with the spacing between the steps in excellent agreement with the D(S=)()(6) value determined by EPR. Analysis of high-resolution optical data (MCD) allows us to determine the single-ion g values and D value (= -1.035 cm(-)(1)) of the constituent Cr(III) ions directly. A vector coupling analysis demonstrates that the cluster ZFS is almost entirely due to the single-ion component. Thus, the relative orientations of the local and cluster magnetic axes can lead to a cluster ZFS of opposite sign to the single-ion value, even when this is the only significant contribution.