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Funding: AG acknowledges the UK-MRBT-CDT funded by EPSRC (EP/J500045/1), BEB acknowledges the Wellcome Trust Institutional Strategic Support fund (204821/Z/16/Z), JET acknowledges the Leverhulme Trust (Early Career Fellowship; ECF-2014-005). Nitroxides are a unique class of persistent radicals finding a wide range of applications, from spin probes to polarizing agents, and recently bis-nitroxides have been used as proof-of-concept molecules for quantum information processing. Here we present the syntheses of pyrroline-based nitroxide (NO) radicals and give a comparision of two possible synthetic routes to form two key intermediates, namely 2,2,5,5-tetramethylpyrroline-1-oxyl-3-acetylene (TPA) and 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylic acid (TPC). TPC and TPA were then used as precursors for the synthesis of three model compounds featuring two distant NO groups with a variable degree of conjugation and thus electronic communication between them. Using relatively facile synthetic routes, we produced a number of mono- and bis-nitroxides with the structures of multiple compounds unambiguously characterized by X-ray crystallography, while Continuous Wave Electron Paramagnetic Resonance (CW-EPR) allowed us quantify the electronic communication in the bis-nitroxides. Our study expands the repertoire of mono- and bis-nitroxides with possibilities of exploiting them for studying quantum coherence effects and as polarizing agents. Publisher PDF

Funding: For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Accepted Author Manuscript version arising. The authors thank the StAnD (St Andrews and Dundee) EPR group for long-standing support and in particular Dr El Mkami for assistance with PDS experiments. J. L.W. acknowledges support by the BBSRC DTP Eastbio (BB/M010996/1). A. G. acknowledges the EPSRC-funded Centre for Doctoral Training in ‘integrated magnetic resonance’, iMR-CDT (EP/J500045/1) B. E. B. and K. A. acknowledge support by the Leverhulme Trust (RPG-2018-397). B. E. B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). Pulse dipolar spectroscopy (PDS) measurements are an important complementary tool in structural biology and are increasingly applied to macromolecular assemblies implicated in human health and disease at physiological concentrations. This requires ever higher sensitivity, and recent advances have driven PDS measurements into the mid-nanomolar concentration regime, though optimization and acquisition of such measurements remains experimentally demanding and time expensive. One important consideration is that constant-time acquisition represents a hard limit for measurement sensitivity, depending on the maximum measured distance. Determining this distance a priori has been facilitated by machine-learning structure prediction (AlphaFold2 and RoseTTAFold) but is often confounded by non-representative behaviour in frozen solution that may mandate multiple rounds of optimization and acquisition. Herein, we endeavour to simultaneously enhance sensitivity and streamline PDS measurement optimization to one-step by benchmarking a variable-time acquisition RIDME experiment applied to CuII-nitroxide and CuII-CuII model systems. Results demonstrate marked sensitivity improvements of both 5- and 6-pulse variable-time RIDME of between 2- and 5-fold over the constant-time analogues. Publisher PDF

Hsp90 is an important molecular chaperone that facilitates the maturation of client proteins. It is a homodimer, and its function depends on a conformational cycle controlled by ATP hydrolysis and co-chaperones binding. We explored the binding of co-chaperone Sba1 to yeast Hsp90 (yHsp90) and the associated conformational change of yHsp90 in the pre- and post-ATP hydrolysis states by double electron-electron resonance (DEER) distance measurements. We substituted the Mg(II) cofactor at the ATPase site with paramagnetic Mn(II) and established the binding of Sba1 by measuring the distance between Mn(II) and a nitroxide (NO) spin-label on Sba1. Then, Mn(II)-NO DEER measurements on yHsp90 labeled with NO at the N-terminal domain detected the shift toward the closed conformation for both hydrolysis states. Finally, Mn(II)-Mn(II) DEER showed that Sba1 induced a closed conformation different from those with just bound Mn(II)·nucleotides. Our results provide structural experimental evidence for the binding of Sba1 tuning the closed conformation of yHsp90.

Gd(III) complexes are currently established as spin labels for structural studies of biomolecules using pulse dipolar electron paramagnetic resonance (PD-EPR) techniques. This has been achieved by the availability of medium- and high-field spectrometers, understanding the spin physics underlying the spectroscopic properties of high spin Gd(III) (S=7/2) pairs and their dipolar interaction, the design of well-defined model compounds and optimization of measurement techniques. In addition, a variety of Gd(III) chelates and labeling schemes have allowed a broad scope of applications. In this review, we provide a brief background of the spectroscopic properties of Gd(III) pertinent for effective PD-EPR measurements and focus on the various labels available to date. We report on their use in PD-EPR applications and highlight their pros and cons for particular applications. We also devote a section to recent in-cell structural studies of proteins using Gd(III), which is an exciting new direction for Gd(III) spin labeling.

Gd(III) complexes are currently established as spin labels for structural studies of biomolecules using pulse dipolar electron paramagnetic resonance (PD-EPR) techniques. This has been achieved by the availability of medium- and high-field spectrometers, understanding the spin physics underlying the spectroscopic properties of high spin Gd(III) (S=7/2) pairs and their dipolar interaction, the design of well-defined model compounds and optimization of measurement techniques. In addition, a variety of Gd(III) chelates and labeling schemes have allowed a broad scope of applications. In this review, we provide a brief background of the spectroscopic properties of Gd(III) pertinent for effective PD-EPR measurements and focus on the various labels available to date. We report on their use in PD-EPR applications and highlight their pros and cons for particular applications. We also devote a section to recent in-cell structural studies of proteins using Gd(III), which is an exciting new direction for Gd(III) spin labeling.

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. GdIII -GdIII distances measured by double electron-electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-GdIII were indistinguishable from GdIII -GdIII distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.

AG and CLM were supported by postgraduate fellowships by the EPSRC-funded doctoral training centre ‘integrated magnetic resonance’. This work was supported by the European Union Seventh Framework Programme (PCIG12-GA-2012-334496). The W-band instrument was developed under the U.K. Research Councils Basic Technology Program (grant EP/F039034/1) and the Q-band equipment is supported by the Wellcome Trust (099149/Z/12/Z). 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 and electronic 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. Postprint

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.

JLW is supported by the BBSRC DTP Eastbio. This work was supported by equipment funding through the Wellcome Trust (099149/Z/12/Z) and BBSRC (BB/R013780/1). We gratefully acknowledge ISSF support to the University of St Andrews from The Wellcome Trust. Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent CuII coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (KD) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm CuII KDs under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled CuII–nitroxide systems using a commercial Q‐band EPR instrument. Publisher PDF

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent CuII coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (K D) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm CuII K Ds under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled CuII–nitroxide systems using a commercial Q‐band EPR instrument.

Triarylmethyl (TAM or trityl) radicals are becoming important for measuring distances in proteins and nucleic acids. Here, we report on a new trityl spin label CT02MA, which conjugates to a protein via a redox stable thioether bond. The performance of the new spin label was demonstrated in W-band double electron-electron resonance (DEER) distance measurements on doubly trityl-labelled mutants of immunoglobulin G-binding protein 1 (GB1) and ubiquitin. For both doubly CT02MA-labelled proteins we measured, by applying chirped pump pulse(s), relatively narrow distance distributions, comparable to those obtained with the same protein mutants doubly labelled with BrPy-DO3MA-Gd(iii). We noticed, however, that the sample contained some free CT02MA that was difficult to remove at the purification step. Dual labelling of ubiquitin with one CT02MA tag and one BrPy-DO3MA-Gd(iii) tag was achieved as well and the trityl-Gd(iii) distance distribution was measured, facilitated by the use of a dual mode cavity in combination with a chirped pump pulse. We also measured the Gd(iii)-Gd(iii) distance distribution in this sample, showing that the labelling procedure was not fully selective. Nevertheless, these measurements demonstrate the potential of the high sensitivity Gd(iii)-trityl W-band DEER distance measurements in proteins, which can be further exploited by designing orthogonal Gd(iii)/trityl labelling schemes.

AG was supported by the EPSRC funded Centre for Doctoral Training in ‘integrated magnetic resonance’ (EP/J500045/1). BEB is grateful for funding from the European Union (REA 334496). This work was supported by the EPSRC (EP/M024660/1), the DFG (Schwerpunktprogramm 1601) and a Wellcome Trust multiuser equipment grant [099149/Z/12/Z]. Fundamentally, the stability of coordination complexes and of templated (bio)macromolecular assemblies depends on the thermodynamic and kinetic properties of the intermediates and final complexes formed. Here, we used pulse EPR (electron paramagnetic resonance) spectroscopy to determine the stabilities of nanoscopic assemblies formed between one or two nitroxide spin-labelled tridentate 2,2′:6′,2′′-terpyridine (tpy) ligands and divalent metal ions (FeII, ZnII, CoII and CuII). In three distinct approaches we exploited (a) the modulation depth of pulsed electron–electron double resonance (PELDOR) experiments in samples with increasing metal-to-ligand ratios, (b) the frequencies of PELDOR under broadband excitation using shaped pulses and (c) the distances recovered from well-resolved PELDOR data in fully deuterated solvents measured at 34 GHz. The results demonstrate that PELDOR is highly sensitive to resolving the stability of templated dimers and allows to readily distinguish anti-cooperative binding (for CuII ions) from cooperative binding (for CoII or FeII ions). In the case of paramagnetic ions (CoII and CuII) the use of broadband PELDOR allowed to identify the cooperativity of binding from the time domain and distance data. By using a second labelled tpy ligand and by mixing two homoleptic complexes of the same metal centre we could probe the kinetic stability on a timescale of tens of seconds. Here, tpy complexes of CuII and ZnII were found to be substitutionally labile, CoII showed very slow exchange and FeII was inert under our conditions. Not only do our chemical models allow studying metal–ligand interactions via PELDOR spectroscopy, the design of our study is directly transferable to (bio)macromolecular systems for determining the kinetic and thermodynamic stabilities underpinning (templated) multimerisation. Considering the limited methods available to obtain direct information on the composition and stability of complex assemblies we believe our approach to be a valuable addition to the armoury of methods currently used to study these systems. Postprint

Biomolecular complexes are often multimers fueling the demand for methods that allow unraveling their composition and geometric arrangement. Pulse electron paramagnetic resonance (EPR) spectroscopy is increasingly applied for retrieving geometric information on the nanometer scale. The emerging RIDME (relaxation‐induced dipolar modulation enhancement) technique offers improved sensitivity in distance experiments involving metal centers (e.g. on metalloproteins or proteins labelled with metal ions). Here, a mixture of a spin labelled ligand with increasing amounts of paramagnetic CuII ions allowed accurate quantification of ligand‐metal binding in the model complex formed. The distance measurement was highly accurate and critical aspects for identifying multimerization could be identified. The potential to quantify binding in addition to the high‐precision distance measurement will further increase the scope of EPR applications.

This article was made open access through BIS OA funding. Nanometre distance measurements by pulsed electron−electron double resonance (PELDOR) spectroscopy have become an increasingly important tool in structural biology. The theoretical underpinning of the experiment is well-defined for systems containing two nitroxide spin-labels (spin pairs) however recently experiments have been reported on homo-oligomeric membrane proteins consisting of up to eight spin-labelled monomers. We have explored the theory behind these systems by examining model systems based on multiple spins arranged in rotationally symmetric polygons. The results demonstrate that with a rising number of spins within the test molecule, increasingly strong distortions appear in distance distributions obtained from an analysis based on the simple spin pair approach. These distortions are significant over a range of system sizes and remain so even when random errors are introduced into the symmetry of the model. We present an alternative approach to the extraction of distances on such systems based on a minimisation that properly treats multi-spin correlations. We demonstrate the utility of this approach on a spin-labelled mutant of the heptameric Mechanosensitive Channel of Small Conductance of E. coli. Publisher PDF

Correction for ‘Assessing dimerisation degree and cooperativity in a biomimetic small-molecule model by pulsed EPR’ by K. Ackermann et al., Chem. Commun., 2015, 51, 5257–5260.