Advances in the synthesis of nitroxide radicals for use in biomolecule spin labelling

Funding: UK EPSRC (EP/L022044/1), and the Royal Society for a University Research Fellowship (JEL).EPR spectroscopy is an increasingly useful analytical tool to probe biomolecule structure, dynamic behaviour, and interactions. Nitroxide radicals are the most commonly used radical probe in EPR experiments, and many methods have been developed for their synthesis, as well as incorporation into biomolecules using site-directed spin labelling. In this Tutorial Review, we discuss the most practical methods for the synthesis of nitroxides, focusing on the tunability of their structures, the manipulation of their sidechains into spin labelling handles, and their installation into biomolecules.PostprintPeer reviewe

ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery

Funding: Royal Society for a University Research Fellowship; Wellcome Multi-User Equipment Grant (099149/Z/12/Z) (JEL).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. eLife 2016]. Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogen-deuterium-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.Publisher PDFPeer reviewe

Synthesis of Next Generation Maleimide Radical Labels

This work was funded by EPSRC, Grant Number EP/LO22044/1, a Royal Society University Research Fellowship to JEL , Royal Society Research Grant RG120645 and support from Active Spectrum Inc.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.PostprintPeer reviewe

Iron is a ligand of SecA-like metal-binding domains in vivo

Funding: JEL thanks the Royal Society for a University Research Fellowship and the Wellcome Trust for the Q-band EPR spectrometer (099149/Z/12/Z).The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modelling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.PostprintPeer reviewe

Distance measurement of a noncovalently bound Y@C82 pair with double electron electron resonance spectroscopy

Paramagnetic endohedral fullerenes with long spin coherence times, such as N@C60 and Y@C82, 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 porphyrinbased two-site receptor 1, offering a rigid structure that binds spin-active fullerenes (Y@C82) 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 (DEER) and analysed to give a distance of 4.87 nm with a small distribution of distances

Advances in the synthesis of nitroxide radicals for use in biomolecule spin labelling

EPR spectroscopy is an increasingly useful analytical tool to probe biomolecule structure, dynamic behaviour, and interactions. Nitroxide radicals are the most commonly used radical probe in EPR experiments, and many methods have been developed for their synthesis, as well as incorporation into biomolecules using site-directed spin labelling. In this Tutorial Review, we discuss the most practical methods for the synthesis of nitroxides, focusing on the tunability of their structures, the manipulation of their sidechains into spin labelling handles, and their installation into biomolecules

Tuning the properties of nitroxide spin labels for use in electron paramagnetic resonance spectroscopy through chemical modification of the nitroxide framework

Spin labels containing nitroxyl radicals possess many properties that render them useful for electron paramagnetic resonance (EPR) spectroscopy. This review describes the relationships between the structure and properties of nitroxide spin labels, methods for their synthesis, advances in methods for their incorporation into biomolecules, and selected examples of applications in biomolecule structural investigations. </p

Combined pulsed electron double resonance EPR and molecular dynamics investigations of calmodulin suggest effect of crowding agents on protein structure (dataset)

EPR data set underpinning publicatio

Expanding the Scope of EPR for Structural Studies of Proteins and Peptides (thesis data)

Data pertaining to the thesis include: EPR data in .DSC and .DTA or .par and .spc format. These can be opened as a text file. LC-MS data raw data are not given as would require the Masslynx software for processing, the .dat files are given which can be opened as a text file with the data. CD data are given in .bka form also openable as a text file. The data files are embargoed until 13/09/202