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Double electron electron resonance (DEER) spectroscopy is an important technique to measure distance distributions P ( r ) for studying protein structures and protein-protein interactions. DEER data analysis can at times become challenging due to the lack of a detailed analytical signal expression or numerical methods with rapid computation time. We have derived an analytical expression κ FULL , which includes both the pseudo-secular dipolar coupling (PSDC) and the finite pulse effects, especially important for shorter distances. Analyses of experiments by κ FULL yield accurate and consistent P ( r ) values for three DEER nitroxide-rulers with distances ( r AVG ) in the range of 15 to 32 Å, while the current standard analysis produces erroneous results for r AVG < 20 Å. Computation times for deriving P ( r ) vary between 1 min and 4 min, which is usually much shorter than previous methods that include pseudo-secular and other effects. The expression can be applied to all types of DEER spin probes with little or no modifications.

Tc toxins are pore-forming virulence factors of many pathogenic bacteria. Following pH-induced conformational changes, they perforate the target membrane like a syringe to translocate toxic enzymes into a cell. Although this complex transformation has been structurally well studied, the reaction pathway and the resulting temporal evolution have remained elusive. We used an integrated biophysical approach to monitor prepore-to-pore transition and found a reaction time of ~30 hours for a complete transition. We show two asynchronous general steps of the process, shell opening and channel ejection, with the overall reaction pathway being a slow multistep process involving three intermediates. Liposomes, an increasingly high pH, or receptors facilitate shell opening, which is directly correlated with an increased rate of the prepore-to-pore transition. Channel ejection is a near-instantaneous process which occurs with a transition time of <60 milliseconds. Understanding the mechanism of action of Tc toxins and unveiling modulators of the kinetics are key steps toward their application as biomedical devices or biopesticides.

We report a family of carbon sorbents synthesized by integrating hypergolics with activation reactions on a templated substrate. The materials design leads to nanoporous carbons with a BET area of 4800 m 2 g -1 with an impressive total pore volume of 2.7 cm 3 g -1 . To the best of our knowledge, this BET area value is the highest reported in the literature. Electron spin resonance (ESR) measurements determined the number of radicals in an effort to provide a mechanistic understanding of the formation of ultrahigh surface area carbons. In combination with XPS, we propose a mechanism based on the synergistic effect between rim-based pentagonal rings and carbon radicals, which we believe can be exploited to produce other highly porous carbons. The CO 2 capture capacity of the hyperporous carbon tested under dynamic CO 2 capture conditions was ∼1.25 mmol g -1 versus 0.66 mmol g -1 of a conventionally activated carbon under similar conditions. The CO 2 capture kinetics were extremely fast and reached 99% of the total capacity within 120 s. Lastly, supercapacitor electrodes deliver a high volumetric energy density of ∼60 W h L -1 and a volumetric power density of 1 kW L -1 , which is the highest reported value for activated carbon.

The C-terminal domain of the cellular prion protein (PrP C ) contains two N-linked glycosylation sites, the occupancy of which impacts disease pathology. In this study, we demonstrate that glycans at these sites are required to maintain an intramolecular interaction with the N-terminal domain, mediated through a previously identified copper-histidine tether, which suppresses the neurotoxic activity of PrP C . NMR and electron paramagnetic resonance spectroscopy demonstrate that the glycans refine the structure of the protein's interdomain interaction. Using whole-cell patch-clamp electrophysiology, we further show that cultured cells expressing PrP molecules with mutated glycosylation sites display large, spontaneous inward currents, a correlate of PrP-induced neurotoxicity. Our findings establish a structural basis for the role of N-linked glycans in maintaining a nontoxic, physiological fold of PrP C ., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lysosomal amino acid efflux by proton-driven transporters is essential for lysosomal homeostasis, amino acid recycling, mTOR signaling, and maintaining lysosomal pH. To unravel the mechanisms of these transporters, we focus on cystinosin, a prototypical lysosomal amino acid transporter that exports cystine to the cytosol, where its reduction to cysteine supplies this limiting amino acid for diverse fundamental processes and controlling nutrient adaptation. Cystinosin mutations cause cystinosis, a devastating lysosomal storage disease. Here, we present structures of human cystinosin in lumen-open, cytosol-open, and cystine-bound states, which uncover the cystine recognition mechanism and capture the key conformational states of the transport cycle. Our structures, along with functional studies and double electron-electron resonance spectroscopic investigations, reveal the molecular basis for the transporter's conformational transitions and protonation switch, show conformation-dependent Ragulator-Rag complex engagement, and demonstrate an unexpected activation mechanism. These findings provide molecular insights into lysosomal amino acid efflux and a potential therapeutic strategy., Competing Interests: Declaration of interests P.S., L.D., and X.L. have a provisional patent for the 3H5 variable sequence: Serial No. 63/366,972, entitled “Anti-Human, Cystinosin Antibodies and Methods of Use Thereof.”, (Copyright © 2022 Elsevier Inc. All rights reserved.)

Constitutive activation of the canonical NF-κB signaling pathway is a major factor in Kaposi's sarcoma-associated herpes virus pathogenesis where it is essential for the survival of primary effusion lymphoma. Central to this process is persistent upregulation of the inhibitor of κB kinase (IKK) complex by the virally encoded oncoprotein vFLIP. Although the physical interaction between vFLIP and the IKK kinase regulatory component essential for persistent activation, IKKγ, has been well characterized, it remains unclear how the kinase subunits are rendered active mechanistically. Using a combination of cell-based assays, biophysical techniques, and structural biology, we demonstrate here that vFLIP alone is sufficient to activate the IKK kinase complex. Furthermore, we identify weakly stabilized, high molecular weight vFLIP-IKKγ assemblies that are key to the activation process. Taken together, our results are the first to reveal that vFLIP-induced NF-κB activation pivots on the formation of structurally specific vFLIP-IKKγ multimers which have an important role in rendering the kinase subunits active through a process of autophosphorylation. This mechanism of NF-κB activation is in contrast to those utilized by endogenous cytokines and cellular FLIP homologues., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1-7). GCAPs form dimers that bind to Ca 2+ and confer Ca 2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg 2+ -bound, Ca 2+ -free, Fe 2+ -free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca 2+ -bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe 2+ binding. Nitroxide spin-label probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe 2+ -dependent regulation of cyclase activity in zebrafish photoreceptors.

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Invited for this month's cover picture is the group of Professor Enrica Bordignon at the Ruhr University Bochum. The cover picture shows an artistic view of E. coli cells and two spin-labeled recombinantly produced proteins, which can be inserted into the cells for EPR studies. The primary sequence of the proteins is schematically shown with the one-letter amino acid code, and cysteine residues are functionalized with the two new gem diethyl nitroxide spin labels designed to better sustain the reducing cellular environment. Read the full text of their Full Paper at 10.1002/open.201900119.

The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins' fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem -diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem -dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the "true" biocompatibility of the gem -diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations., Competing Interests: The authors declare no conflict of interest.

Light-exposed organisms developed photoreceptors to transduce light signals for environmental adaptation. Phytochromes, found in bacteria, fungi, and plants, can discriminate the ratio of red and far-red light using the isomerization of a bilin chromophore bound to a photosensory module to trigger downstream conformational changes in the protein. Here, we investigated by hydrogen/deuterium exchange mass spectrometry and electron paramagnetic resonance spectroscopy the light-driven domain mechanics of a minimal monomeric photosensory module from the group II phytochrome Cph2 from Synechocystis sp. PCC 6803. We could unambiguously trace the light-driven secondary structural rearrangement of its tongue region, and we found a translational motion of the PHY domain that is related to what was found before by X-ray studies in a group I module. Our analysis demonstrates a common light response in the photosensory modules of phytochromes, orchestrated solely by the GAF-PHY bidomain independent of further quaternary interactions or the nature of downstream effector domains., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into the outer mitochondrial membrane, oligomerizes, and forms pores that release proapoptotic factors like Cytochrome c into the cytosol. The structures of active Bax and its homolog Bak are only partially understood and the topology of the proteins with respect to the membrane bilayer is controversially described in the literature. Here, we systematically review and examine the protein-membrane, protein-water, and protein-protein contacts of the nine helices of active Bax and Bak, and add a new set of topology data obtained by fluorescence and EPR methods. We conclude based on the consistent part of the datasets that the core/dimerization domain of Bax (Bak) is water exposed with only helices 4 and 5 in membrane contact, whereas the piercing/latch domain is in peripheral membrane contact, with helix 9 being transmembrane. Among the available structural models, those considering the dimerization/core domain at the rim of a toroidal pore are the most plausible to describe the active state of the proteins, although the structural flexibility of the piercing/latch domain does not allow unambiguous discrimination between the existing models.