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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.

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.

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.

ATP-dependent chromatin remodelling proteins represent a diverse family of proteins that share ATPase domains that are adapted to regulate protein–DNA interactions. Here, we present structures of the Saccharomyces cerevisiae Chd1 protein engaged with nucleosomes in the presence of the transition state mimic ADP-beryllium fluoride. The path of DNA strands through the ATPase domains indicates the presence of contacts conserved with single strand translocases and additional contacts with both strands that are unique to Snf2 related proteins. The structure provides connectivity between rearrangement of ATPase lobes to a closed, nucleotide bound state and the sensing of linker DNA. Two turns of linker DNA are prised off the surface of the histone octamer as a result of Chd1 binding, and both the histone H3 tail and ubiquitin conjugated to lysine 120 are re-orientated towards the unravelled DNA. This indicates how changes to nucleosome structure can alter the way in which histone epitopes are presented., eLife digest The DNA inside cells contains all the information needed to build an organism. Human DNA measures about 2 metres. To condense it, DNA is wrapped around eight histone proteins to form disc-like structures, called nucleosomes. Nucleosomes are further compressed into chromatin fibres that make up our chromosomes. The way DNA is packaged and positioned into the nucleosomes can be variably controlled and affects how genes are switched on and off. Although all cells have the same DNA, the way specific genes are turned on and off gives rise to the different types of cells in our body. Specialised motor proteins, called ‘chromatin remodellers’, control the positioning of nucleosomes inside cells. In yeast cells, for example, the protein Chd1 moves nucleosomes along the DNA so that they are evenly spaced. So far, it has been unclear how chromatin remodellers interact with nucleosomes. To investigate this further, Sundaramoorthy et al. studied the structure of Chd1 bound to a nucleosome, in which one histone protein was modified with a molecule, called ubiquitin, which is present on genes where Chd1 is known to be active. The structure revealed that both Chd1 and the nucleosome did not have their usual shape. Moreover, Chd1 partially unwrapped DNA from the nucleosomes. As a consequence, the ubiquitin moved to interact with the unwrapped DNA; as did a flexible area on one of the histones, known as ‘histone tail’. Both ubiquitin and histone tails play important roles in signalling processes on chromatin. Therefore, such a rearrangement could affect the transmission of signals from chromatin. The organisation of nucleosomes affects the accessibility of the underlying DNA. As a result, any process that happens on DNA is affected – including controlling when genes are turned on and off under normal conditions, and when things go wrong during diseases. A better knowledge of how the organisation of nucleosomes is controlled will improve our understanding of gene regulation.

Claire Motion would like to acknowledge funding from EPSRC as part of the iMR-CDT. Stacey Bell and Janet Lovett thank EPSRC Grant Number EP/LO22044/1. Janet Lovett also thanks the Royal Society for a University Research Fellowship and Research Grant RG120645. Sabine Van Doorslaer acknowledges the Research Foundation – Flanders (FWO) for financial support (grant G.0687.13). The W-band instrument was developed under the UK Research Council’s Basic Technology Program (grant EP/F039034/1). We also thank the Wellcome Trust (grant 099149/Z/12/Z). This work demonstrates the feasibility of making sensitive nanometer distancemeasurements 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 30times 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. Publisher PDF

M.A.S. & J.E.M. would like to acknowledge funding from the EPSRC as part of the iMR-CDT. The Authors would like to acknowledge funding from The MRC UK, Grant G1100021, EPSRC Basic Technology EP/F039034/1, and from the Wellcome Trust 099149/Z/12/Z. The bipedal spin label Rx is more restricted in its conformation and dynamics than its monopodal counterpart R1. To systematically investigate the utility of the Rx label, we have attempted to comprehensively survey the attachment of Rx to protein secondary structures. We have examined the formation, structure and dynamics of the spin label in relation to the underlying protein in order to determine feasibility and optimum conditions for distance and orientation measurement by pulsed EPR. The labeled proteins have been studied using molecular dynamics, CW EPR, pulsed EPR distance measurement at X-band and orientation measurement at W-band. The utility of different modes and positions of attachment have been compared and contrasted. Publisher PDF