Cryogenic and Dissolution DNP NMR on γ-Irradiated Organic Molecules.

Nuclear magnetic resonance (NMR) plays a central role in the elucidation of chemical structures but is often limited by low sensitivity. Dissolution dynamic nuclear polarization (dDNP) emerges as a transformative methodology for both solution-state NMR and metabolic NMR imaging, which could overcome this limitation. Typically, dDNP relies on combining a stable radical with the analyte within a uniform glass under cryogenic conditions. The electron polarization is then transferred through microwave irradiation to the nuclei. The present study explores the use of radicals introduced via γ-irradiation, as bearers of the electron spins that will enhance 1H or 13C nuclides. 1H solid-state NMR spectra of γ-irradiated powders at 1-5 K revealed, upon microwave irradiation, signal enhancements that, in general, were higher than those achieved through conventional glass-based DNP. Transfer of these samples to a solution-state NMR spectrometer via a rapid dissolution driven by a superheated water provided significant enhancements of solution-state 1H NMR signals. Enhancements of 13C signals in the γ-irradiated solids were more modest, as a combined consequence of a low radical concentration and of the dilute concentration of 13C in the natural abundant samples examined. Nevertheless, ca. 700-800-fold enhancements in 13C solution NMR spectra of certain sites recorded at 11.7 T could still be achieved. A total disappearance of the radicals upon performing a dDNP-like aqueous dissolution and a high stability of the samples were found. Overall, the study showcases the advantages and limitations of γ-irradiated radicals as candidates for advancing spectroscopic dDNP-enhanced NMR.

C-terminal domain dimerization in yeast Hsp90 is moderately modulated by the other domains.

Heat shock protein 90 (Hsp90) serves as a crucial regulator of cellular proteostasis by stabilizing and regulating the activity of numerous substrates, many of which are oncogenic proteins. Therefore, Hsp90 is a drug target for cancer therapy. Hsp90 comprises three structural domains, a highly conserved amino-terminal domain (NTD), a middle domain (MD), and a carboxyl-terminal domain (CTD). The CTD is responsible for protein dimerization, is crucial for Hsp90's activity, and has therefore been targeted for inhibiting Hsp90. Here we addressed the question of whether the CTD dimerization in Hsp90, in the absence of bound nucleotides, is modulated by allosteric effects from the other domains. We studied full length (FL) and isolated CTD (isoC) yeast Hsp90 spin-labeled with a Gd(III) tag by double electron-electron resonance measurements to track structural differences and to determine the apparent dissociation constant (Kd). We found the distance distributions for both the FL and isoC to be similar, indicating that the removal of the NTD and MD does not significantly affect the structure of the CTD dimer. The low-temperature double electron-electron resonance-derived Kd values, as well as those obtained at room temperature using microscale thermophoresis and native mass spectrometry, collectively suggested the presence of some allosteric effects from the NTDs and MDs on the CTD dimerization stability in the apo state. This was evidenced by a moderate increase in the Kd for the isoC compared with the FL mutants. Our results reveal a fine regulation of the CTD dimerization by allosteric modulation, which may have implications for drug targeting strategies in cancer therapy.

Enhanced sensitivity for pulse dipolar EPR spectroscopy using variable-time RIDME.

Pulse dipolar EPR 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.

Synthesis of mono-nitroxides and of bis-nitroxides with varying electronic through-bond communication.

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.

Monitoring the Conformation of the Sba1/Hsp90 Complex in the Presence of Nucleotides with Mn(II)-Based Double Electron-Electron Resonance.

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.

Characteristics of Gd(III) spin labels for the study of protein conformations.

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.

Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging.

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.

Two closed ATP- and ADP-dependent conformations in yeast Hsp90 chaperone detected by Mn(II) EPR spectroscopic techniques.

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.

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 KD s 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.

DEER distance measurements on trityl/trityl and Gd(iii)/trityl labelled proteins.

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.

Small neutral Gd(iii) tags for distance measurements in proteins by double electron-electron resonance experiments.

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.

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

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 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.

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.

PELDOR in rotationally symmetric homo-oligomers.

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.