Copper homeostasis is essential for mammalian physiology. Serum albumin plays an important role in plasma copper transport and buffering, yet its Cu2+ binding sites have remained incompletely characterised. Here we report the first X-ray crystal structure of a mammalian serum albumin, in this case equine albumin, bound to copper(ii). The structure revealed a high-affinity ATCUN site with characteristic square-planar geometry. Additional Cu2+ binding was observed at five secondary sites, including sites A and B and other histidine-containing sites (involving either His287, His317 and His509). Continuous-wave EPR spectroscopy further supported a square-planar coordination at the ATCUN site through a low-g spectral feature appearing upon binding of less than one molar equivalent of Cu2+. ESEEM and HYSCORE experiments detected nuclear quadrupole interactions and weakly coupled 14N signals, supporting histidine involvement and increased water coordination at higher Cu2+ loading. RIDME-derived distance distributions and structural simulations indicate simultaneous occupancy of multiple sites, with strong evidence for ATCUN and site B, and partial engagement of distal histidines (His287, His317) at elevated Cu2+ equivalents. These findings support a dynamic, multi-site binding model in which short-range distances arise from ATCUN and site B co-occupancy, while longer-range peaks reflect contributions from distal histidine sites.

The potassium two-pore domain (K2P) ion channel TRAAK is expressed in the nervous system and regulates the fast action potential in membranes. Like all K2P channels, TRAAK possesses a distinct extracellular cap, which adopts a swapped and a non-swapped conformation1,2. However, the proportional representation of these two species within native membranes - and the trigger or stimulus associated with this conformational transition - are unknown. Here, we utilise Pulse Dipolar EPR Spectroscopy combined with heterologous single-subunit spin-labelling and monitor the complete conformational ensemble of TRAAK's cap domain in membranes. We demonstrate the coexistence of the swapped and non-swapped states, quantify their populations within the TRAAK ensemble, and show that the swapped conformation dominates, with the ratio being influenced by temperature. Native lipid analysis shows that TRAAK selectively associates with and is activated by signalling lipids to the exclusion of membrane-dominant phosphatidylcholine lipids from its vicinity, forming a distinct microdomain. Our approach can identify the immediate lipid environment, detect and quantify cap state populations in homo-/hetero-K2P channels and link domain swapping to specific triggers.

In-cell electron paramagnetic resonance (EPR) spectroscopy requires robust spin-labelling strategies compatible with cellular conditions. Cu II -NTA coordination to genetically engineered double-histidine (dHis) motifs has shown promise for endogenous labelling in E. coli . Here, we evaluated the effect of varying Cu II -NTA affinity on the success of these experiments. Relaxation induced dipolar modulation enhancement (RIDME)-based titrations revealed dissociation constants ( K d ) in the 10 -6 and 10 -8 range for two different β-sheet i and i+2 dHis sites. In-cell EPR spectra demonstrated that the persistence of the Cu II EPR signal correlates with these binding affinities. The success of in-cell pulsed electron-electron double resonance (PELDOR) involving both β-sheet sites and a high-affinity α-helical site depended on the site; the higher-affinity site yielded analyzable results, the lower-affinity site did not. These results highlight the critical importance of low K d binding sites for reliable in-cell distance measurements with endogenous Cu II -NTA labelling and the substantial sensitivity gain offered by RIDME experiments.

In-cell electron paramagnetic resonance (EPR) spectroscopy requires robust spin-labeling strategies compatible with cellular conditions. CuII-NTA coordination to genetically engineered double-histidine (dHis) motifs has shown promise for endogenous labeling in Escherichia coli. Here, we evaluated the effect of varying CuII-NTA affinity on the success of these experiments. Relaxation induced dipolar modulation enhancement (RIDME)-based titrations revealed dissociation constants (Kd) in the 10-6 and 10-8 range for two different β-sheet i and i+2 dHis sites. In-cell EPR spectra demonstrated that the persistence of the CuII EPR signal correlates with these binding affinities. The success of in-cell pulsed electron-electron double resonance (PELDOR) involving both β-sheet sites and a high-affinity α-helical site depended on the site; the higher-affinity site yielded analyzable results, whereas the lower-affinity site did not. These results highlight the critical importance of low Kd binding sites for reliable in-cell distance measurements with endogenous CuII-NTA labeling and the substantial sensitivity gain offered by RIDME experiments.

M proteins are major virulence factors of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subspecies equisimilis (SDSE), which have seen a recent surge in invasive infections on a global scale. Despite the importance of M proteins in the pathogenicity of bacteria, they have eluded high-resolution structure determination in their full-length physiologically relevant form, and little is known about their structure and functional dynamics. The publication of AlphaFold and particularly AlphaFold 3 has opened hitherto unknown possibilities for techniques complementing high-resolution structure determination. Electron paramagnetic resonance (EPR) spectroscopy is well-suited for experimental protein structure validation. Here, we demonstrated this confirming a highly unusual N-terminal fold in the full-length M3 protein known to induce autoimmune rheumatic disease in humans.

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy (PDS) is a powerful method for determining nanometer distances in biomolecular systems. This methodology has been successfully used to validate structure hypotheses and to identify disorder. However, inherent distance limitations are under-explored. Many challenging biomolecular applications require long distance information, driving the need for a benchmarking study. We demonstrate distances up to 10.5 nm in TRIM25 reliably extracted from data obtained under standard sample conditions. By protein perdeuteration accurate distance measurements up to 17.3 nm were enabled. Enhancing sensitivity through optimization of the measurement and sample preparation conditions the achievable dipolar evolution time was extended to 150 µs. Thus, distances close to 20 nm should become experimentally accessible, highlighting the potential of advanced PDS methodologies for probing previously inaccessible long-range structural information in complex systems.

Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the non-covalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, double electron-electron resonance (DEER) and relaxation-induced dipolar modulation enhancement (RIDME), were simultaneously employed for studying 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 cooperativity of protein dimerization across α-helical double-histidine motifs in presence of Cu(II) is strongly buffer modulated, 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.

Cyclic dipeptides are produced by organisms across all domains of life, with many exhibiting anticancer and antimicrobial properties. Oxidations are often key to their biological activities, particularly C-C bond oxidation catalysed by tailoring enzymes including cyclodipeptide oxidases. These flavin-dependent enzymes are underexplored due to their intricate three-dimensional arrangement involving multiple copies of two distinct small subunits, and mechanistic details underlying substrate selection and catalysis are lacking. Here, we determined the structure and mechanism of the cyclodipeptide oxidase from the halophile Nocardiopsis dassonvillei (NdasCDO), a component of the biosynthetic pathway for nocazine natural products. We demonstrated that NdasCDO forms filaments in solution, with a covalently bound flavin mononucleotide (FMN) cofactor at the interface between three distinct subunits. The enzyme exhibits promiscuity, processing various cyclic dipeptides as substrates in a distributive manner. The reaction is optimal at high pH and involves the formation of a radical intermediate. Pre-steady-state kinetics, a significant solvent kinetic isotope effect, and the absence of viscosity effects suggested that a step linked to FMN regeneration controlled the reaction rate. Our work elucidates the complex mechanistic and structural characteristics of this dehydrogenation reaction, positioning NdasCDO as a promising biocatalyst and expanding the FMN-dependent oxidase family to include enzyme filaments.

Human serum albumin (HSA) is the most abundant plasma protein, which functions to transport a large range of ligands within the circulation. These interactions have important implications for human health and disease. The primary binding site for CuII ions on HSA is known to be the so-called amino-terminal CuII and NiII binding (ATCUN) motif. However, the number and identity of secondary binding sites is currently not understood. In this study, we harnessed a suite of contemporary electron paramagnetic resonance (EPR) spectroscopy methods to investigate recombinantly produced constructs of HSA bearing single-histidine knockouts, with the aim to characterise its endogenous CuII ion binding sites.

Riboswitches control gene regulation upon external stimuli such as environmental factors or ligand binding. The fluoride sensing riboswitch from Thermotoga petrophila is a complex regulatory RNA proposed to be involved in resistance to F- cytotoxicity. The details of structure and dynamics underpinning the regulatory mechanism are currently debated. Here we demonstrate that a combination of pulsed electron paramagnetic resonance (ESR/EPR) spectroscopies, detecting distances in the angstrom to nanometre range, can probe distinct regions of conformational flexibility in this riboswitch. PELDOR (pulsed electron-electron double resonance) revealed a similar preorganisation of the sensing domain in three forms, i.e. the free aptamer, the MgII-bound apo, and the F--bound holo form. 19F ENDOR (electron-nuclear double resonance) was used to investigate the active site structure of the F--bound holo form. Distance distributions without a priori structural information were compared with in silico modelling of spin label conformations based on the crystal structure. While PELDOR, probing longer distances, revealed varying conformational flexibility of the RNA backbone, ENDOR indicated low structural heterogeneity at the ligand binding site. Overall, the combination of PELDOR and ENDOR with sub-angstrom precision gave insight into structural organisation and flexibility of a riboswitch, not easily attainable by other biophysical techniques.

Recent sensitivity enhancements in pulse dipolar EPR spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science, as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin labelling using copper(II) ions and trityl yields a more than 3-fold sensitivity increase compared to the established copper(II)-nitroxide labelling strategy. Application of the recently developed variable-time RIDME method yields a further approximately 2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, more than two times lower than previously reported. We expect that experiments at single digit nanomolar concentrations are imminent, which has the potential to transform biological PDS applications.

CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

Membrane forces shift the equilibria of mechanosensitive channels enabling them to convert mechanical cues into electrical signals. Molecular tools to stabilize and methods to capture their highly dynamic states are lacking. Cyclodextrins can mimic tension through the sequestering of lipids from membranes. Here we probe the conformational ensemble of MscS by EPR spectroscopy, the lipid environment with NMR, and function with electrophysiology under cyclodextrin-induced tension. We show the extent of MscS activation depends on the cyclodextrin-to-lipid ratio, and that lipids are depleted slower when MscS is present. This has implications in MscS’ activation kinetics when distinct membrane scaffolds such as nanodiscs or liposomes are used. We find MscS transits from closed to sub-conducting state(s) before it desensitizes, due to the lack of lipid availability in its vicinity required for closure. Our approach allows for monitoring tension-sensitive states in membrane proteins and screening molecules capable of inducing molecular tension in bilayers.

Numerous hormones and genes exhibit diurnal 24-hr rhythms that can also be affected by sleep deprivation. Here we studied diurnal rhythms in DNA methylation under a 24-hr sleep/wake cycle and a subsequent 29 hr of continual wakefulness (1 night of sleep deprivation). Fifteen healthy men (19-35 years) spent 3 days/nights in a sleep laboratory: (1) adaptation; (2) baseline; (3) total sleep deprivation day/night. DNA methylation was analysed from peripheral blood leukocytes, collected every 3 hr for 45 hr (starting at 15:00 hours) during the baseline period and the total sleep deprivation period. Epigenome-wide DNA methylation variation was assessed with the Infinium MethylationEPIC v2.0 Beadchip kit. Rhythm analysis was performed separately for the baseline and the total sleep deprivation time-series data. Pairwise analysis between diurnal samples and sleep deprivation samples at the same timepoint was also carried out to detect differentially methylated positions related to sleep deprivation. Of all DNA methylation sites, 14% exhibited a diurnal rhythm in methylation on the baseline day/night that was altered by sleep deprivation. During sleep deprivation, the number of differentially methylated positions increased towards the end of the sleep deprivation period, with a dominating pattern of hypomethylation. Among differentially methylated positions, an enrichment of genes related to the FAS immune response pathway was detected. In conclusion, DNA methylation exhibits diurnal rhythmicity, and this time-of-day variation needs to be considered when studying DNA methylation as a biomarker in biomedical studies. In addition, the observed DNA methylation changes under wakefulness might serve as a mediator of sleep deprivation-related immune response alterations.

Biomolecular applications of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) are becoming increasingly valuable in structural biology. Site-directed spin labelling of proteins is routinely performed using nitroxides, with paramagnetic metal ions and other organic radicals gaining popularity as alternative spin centres. Spectroscopically orthogonal spin labelling using different types of labels potentially increases the information content available from a single sample. When analysing experimental distance distributions between two nitroxide spin labels, the site-specific rotamer information has been projected into the distance and is not readily available, and the contributions of individual labelling sites to the width of the distance distribution are not obvious from the PDS data. Here, we exploit the exquisite precision of labelling double-histidine (dHis) motifs with Cu II chelate complexes. The contribution of this label to the distance distribution widths in model protein GB1 has been shown to be negligible. By combining a dHis Cu II labelling site with cysteine-specific nitroxide labelling, we gather insights on the label rotamers at two distinct sites, comparing their contributions to distance distributions based on different in silico modelling approaches and structural models. From this study, it seems advisable to consider discrepancies between different in silico modelling approaches when selecting labelling sites for PDS studies.

Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.

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 to 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.

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.