Strategies for synthesis of yardsticks and abaci for nanometre distance measurements by pulsed EPR

Silvia Valera is grateful for support by EPSRC and Bela E. Bode acknowledges support by EastCHEM.Pulsed electron paramagnetic resonance (EPR) techniques have been found to be an efficient tool for elucidation of structure in complex biological systems as they give access to distances in the nanometre range. These measurements can provide additional structural information such as relative orientations, structural flexibility or aggregation states. A wide variety of model systems for calibration and optimisation of pulsed experiments has been synthesised. Their design is based on mimicking biological systems or materials in specific properties such as the distances themselves and the distance distributions. Here, we review selected approaches to the synthesis of chemical systems bearing two or more spin centres, such as nitroxide or trityl radicals, metal ions or combinations thereof and sketch their application in pulsed EPR distance measurements.Publisher PDFPeer reviewe

Verbi in serie: una prospettiva tipologica

The authors acknowledge EPSRC (EP/K503162/1) for funding this research.Na2MoO2−δF4+δ (δ ∼ 0.08) displays a unique variant of the perovskite structure, with simultaneous (Na,vacancy) ordering on the A-site, (Na,Mo) ordering on the B-site, (O,F) ordering on the anion site and an unusual NaNbO3-like octahedral tilt system.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

Pulse dipolar electron paramagnetic resonance spectroscopy reveals buffer modulated cooperativity of metal templated protein dimerization

Funding: Leverhulme Trust - RPG-2018397; Biotechnology and Biological Sciences Research Council - BB/M010996/1; Engineering and Physical Sciences Research Council - EP/N509759/1; Wellcome Trust - 204821/Z/16/Z.Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the noncovalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, pulse electron–electron double resonance and relaxation-induced dipolar modulation enhancement, were simultaneously employed to study the CuII-templated dimerization behavior of a model protein (Streptococcus sp. group G, protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate that the cooperativity of protein dimerization across α-helical double-histidine motifs in the presence of CuII is strongly modulated by the buffer, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.Publisher PDFPeer reviewe

Diphosphane 2,2′-binaphtho[1,8-de][1,3,2]dithiaphosphinine and the easy formation of a stable phosphorus radical cation

A convenient synthesis route to 2,2’-binaphtho[1,8-de][1,3,2]di-thiaphosphinine (3) was found. Its stable radical cation 3•+ was accessed easily through one-electron oxidation with NOBF4.PostprintPostprintPeer reviewe

Investigating native metal ion binding sites in mammalian histidine-rich glycoprotein

Funding: For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) license to any Accepted Author Manuscript version arising. They acknowledge support by the Wellcome Trust (204821/Z/16/Z), the British Heart Foundation (PG/15/9/31270 and FS/15/42/31556), and the Leverhulme Trust (RPG-2018–397). J.L.W. acknowledges support by the BBSRC DTP Eastbio. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1).Mammalian histidine-rich glycoprotein (HRG) is a highly versatile and abundant blood plasma glycoprotein with a diverse range of ligands that is involved in regulating many essential biological processes, including coagulation, cell adhesion, and angiogenesis. Despite its biomedical importance, structural information on the multi-domain protein is sparse, not least due to intrinsically disordered regions that elude high-resolution structural characterization. Binding of divalent metal ions, particularly ZnII, to multiple sites within the HRG protein is of critical functional importance and exerts a regulatory role. However, characterization of the ZnII binding sites of HRG is a challenge; their number and composition as well as their affinities and stoichiometries of binding are currently not fully understood. In this study, we explored modern electron paramagnetic resonance (EPR) spectroscopy methods supported by protein secondary and tertiary structure prediction to assemble a holistic picture of native HRG and its interaction with metal ions. To the best of our knowledge, this is the first time that this suite of EPR techniques has been applied to count and characterize endogenous metal ion binding sites in a native mammalian protein of unknown structure.Publisher PDFPeer reviewe

Understanding the structure directing action of copper-polyamine complexes in the direct synthesis of Cu-SAPO-34 and Cu-SAPO-18 catalysts for the selective catalytic reduction of NO with NH3

This work has been supported by Johnson Matthey PLC, UK.Cu2+ cations complexed by linear polyamines have been studied as structure-directing agents (SDAs) for the direct synthesis of copper-containing microporous silicoaluminophosphate (SAPO) materials. The complexing ligands diethylenetriamine (DETA), N-(2-hydroxyethyl)ethylenediamine (HEEDA), triethylenetetramine (TETA), N,N′-bis(2-aminoethyl)-1,3-propanediamine (232), 1,2-bis(3-aminopropylamino)ethane (323), tetraethylenepentamine (TEPA) and pentaethylenehexamine (PEHA) have been investigated. For comparison, syntheses have been performed using the analogous nickel-polyamine complexes. Cu2+ and Ni2+ forms of both SAPO-18 and SAPO-34 materials have been prepared. While most polyamine complexes direct crystallisation to SAPO-34, SAPO-18 has been prepared with Cu2+(232), Ni2+(232) and Ni2+(TETA). The coordination geometry of the included metal complexes was studied by UV-visible and EPR spectroscopy and computer simulation. SAPO-18 is favoured by the smaller square planar complexes or octahedral species (with 2 water molecules) of 232 and TETA. Calcination leaves extra-framework Cu2+ and Ni2+ cations within SAPO-18 and SAPO-34 frameworks. In situ synchrotron IR spectroscopy of Ni-SAPO-18 has shown thermal template degradation occurs via nitrile intermediates. Rietveld structural analysis located extra-framework Cu2+ and Ni2+ cations released by calcination. In SAPO-34, Cu2+ and Ni2+ were located in the 8R window of the cha cage. A second site was found for Ni2+ at the centre of the six-membered rings (6Rs) of the double-six-ring (D6R) sub-units. In SAPO-18 both Cu2+ and Ni2+ cations were located only in the 6Rs of the D6R sub-units. Selected copper SAPO-18 and SAPO-34 samples were tested in the selective catalytic reduction of NO with ammonia (NH3-SCR); both showed high activity.PostprintPostprintPeer reviewe

Antiviral signaling by a cyclic nucleotide activated CRISPR protease

Funding information: M.G. and J.L.S.B. are funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy–EXC2151–390873048. M.F.W. acknowledges a European Research Council Advanced Grant (grant number 101018608) and the China Scholarship Council (REF: 202008420207 to H.C.). G.H. is grateful for funding by the Deutsche Forschungsgemeinschaft (grant number HA6805/6-1).CRISPR defense systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition3-5. Among a large set of proteins that were linked to type III systems and predicted to bind cOAs6,7, a CRISPR associated Lon protease (CalpL) stood out to us. The protein contains a sensor domain of the SAVED (SMODS-associated and fused to various effector domains) family7, fused to a Lon protease effector domain. However, the mode of action of this effector was unknown. Here, we report the structure and function of CalpL and show that the soluble protein forms a stable tripartite complex with two further proteins, CalpT and CalpS, that are encoded in the same operon. Upon activation by cA4, CalpL oligomerizes and specifically cleaves the MazF-homolog CalpT, releasing the extracytoplasmic function (ECF) sigma factor CalpS from the complex. This provides a direct connection between CRISPR-based foreign nucleic acid detection and transcriptional regulation. Furthermore, the presence of a cA4-binding SAVED domain in a CRISPR effector reveals an unexpected link to the cyclic oligonucleotide-based antiphage signaling system (CBASS).PostprintPeer reviewe

Hexakis{4-[(4'-hydroxybiphenyl-4-yl)ethynyl]phenyl}benzene

A novel polyphenolic backbone, hexakis{4-[(4'-hydroxybiphenyl-4-yl)ethynyl]phenyl}benzene, was synthesised using a common synthetic protocol used for the synthesis of similar polyphenolic compounds.Publisher PDFPeer 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