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 Qband 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.

Transport of proteins across membranes is a fundamental process, achieved in every cell by the ‘Sec’ translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.

Paramagnetic endohedral fullerenes with long spin coherence times, such as N@C and Y@C, are being explored as potential spin quantum bits (qubits). Their use for quantum information processing requires a way to hold them in fixed spatial arrangements. Here we report the synthesis of a porphyrin-based two-site receptor 1, offering a rigid structure that binds spin-active fullerenes (Y@C) at a center-to-center distance of 5.0 nm, predicted from molecular simulations. The spin-spin dipolar coupling was measured with the pulsed EPR spectroscopy technique of double electron electron resonance and analyzed to give a distance of 4.87 nm with a small distribution of distances.

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The synthesis and characterization of four new nitroxide-radical containing next generation maleimides are presented. Each new label has a single leaving group which is either a phenoxyl or bromide. The linker between the maleimide and the nitroxide-containing framework is either a racemic mixture of a short chain or an achiral longer chain. These molecules have been designed to site-specifically label vicinal cysteines in proteins for magnetic resonance studies. The characterization of the final products includes crystallography and the labeling of Sperm Whale Myoglobin protein.Key words Next generation maleimide, succinimide, nitroxide spin label, radical, EPR spectroscopy, PRE NMR, cysteine labeling, site directed spin labeling.Spin labels are small molecules that attach to larger molecules of interest and that stabilize a free radical. The spin labels are most likely to be used to report on their microenvironment, accessibility, motional dynamics or to enable nanometre-scale distance measurements by electron paramagnetic resonance (EPR) spectroscopy. 3 Alternatively, they may be used as paramagnetic relaxation enhancing reagents in nuclear magnetic resonance (NMR) spectroscopy. 4 It is therefore often desirable to have a spin label that demonstrates little conformational freedom while having enough flexibility to avoid disruption of the protein fold.Commonly these labels stabilize a nitroxyl radical as part of a 5 or 6 membered carbon framework and are hence called nitroxide spin labels. These are functionalized to react at the site of interest. For proteins this site is often the thiol of cysteine. The labels can be made to covalently attach reversibly or irreversibly with short or long linkers. Attachment chemistries

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