Monitoring complex formation by relaxation-induced pulse electron paramagnetic resonance distance measurements

Funding: EPSRC DTC and Wellcome (099149/Z/12/Z).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.Publisher PDFPeer reviewe

A low-spin CoII/Nitroxide complex for distance measurements at Q-band frequencies

Funding: AG was supported by the EPSRC funded Centre for Doctoral Training in ‘integrated magnetic resonance’, iMR-CDT (EP/J500045/1) the time the research was conducted. BEB is supported by the Leverhulme Trust (RPG-2018–397) and equipment funding from BBSRC (BB/R013780/1 and BB/T017740/1).Pulse dipolar electron paramagnetic resonance spectroscopy (PDS) is continuously furthering the understanding of chemical and biological assemblies through distance measurements in the nanometer range. New paramagnets and pulse sequences can provide structural insights not accessible through other techniques. In the pursuit of alternative spin centers for PDS, we synthesized a low-spin CoII complex bearing a nitroxide (NO) moiety, where both the CoII and NO have an electron spin S of 1/2. We measured CoII-NO distances with the well-established double electron–electron resonance (DEER aka PELDOR) experiment, as well as with the five- and six-pulse relaxation-induced dipolar modulation enhancement (RIDME) spectroscopies at Q-band frequencies (34 GHz). We first identified challenges related to the stability of the complex in solution via DEER and X-ray crystallography and showed that even in cases where complex disproportionation is unavoidable, CoII-NO PDS measurements are feasible and give good signal-to-noise (SNR) ratios. Specifically, DEER and five-pulse RIDME exhibited an SNR of ~100, and while the six-pulse RIDME exhibited compromised SNR, it helped us minimize unwanted signals from the RIDME traces. Last, we demonstrated RIDME at a 10 μM sample concentration. Our results demonstrate paramagnetic CoII to be a feasible spin center in medium magnetic fields with opportunities for PDS studies involving CoII ions.Publisher PDFPeer reviewe

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

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.PostprintPeer reviewe

Sub-micromolar pulse dipolar EPR spectroscopy reveals increasing CuII-labelling of double-histidine motifs with lower temperature

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 PDFPeer reviewe

Nitroxide-nitroxide and nitroxide-metal distance measurements in transition metal complexes with two or three paramagnetic centres give access to thermodynamic and kinetic stabilities

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.PostprintPeer reviewe

PELDOR in rotationally symmetric homo-oligomers

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 PDFPeer reviewe

Assessing dimerisation degree and cooperativity in a biomimetic small-molecule model by pulsed EPR

AG acknowledges a postgraduate fellowship by the EPSRC funded doctoral training centre ‘integrated magnetic resonance’. BEB is grateful for an EaStCHEM Hirst Academic Fellowship by the School of Chemistry, St Andrews, and funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (REA 334496).Pulsed electron paramagnetic resonance (EPR) spectroscopy is gaining increasing importance as a complementary biophysical technique in structural biology. Here, we describe the synthesis, optimisation, and EPR titration studies of a spin-labelled terpyridine Zn(II) complex serving as a small-molecule model system for tuneable dimerisation.Publisher PDFPeer reviewe

Monitoring complex formation by relaxation-induced pulse electron paramagnetic resonance distance measurements

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