The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex.

The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of ß-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.

While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Escherichia coli Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C'-15N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra.

Electron and Spin Delocalization in [Co6 Se8 (PEt3 )6 ]0/+1 Superatoms.

Molecular clusters can function as nanoscale atoms/superatoms, assembling into superatomic solids, a new class of solid-state materials with designable properties through modifications on superatoms. To explore possibilities on diversifying building blocks, here we thoroughly studied one representative superatom, Co6 Se8 (PEt3 )6 . We probed its structural, electronic, and magnetic properties and revealed its detailed electronic structure as valence electrons delocalize over inorganic [Co6 Se8 ] core while ligands function as an insulated shell. 59 Co SSNMR measurements on the core and 31 P, 13 C on the ligands show that the neutral Co6 Se8 (PEt3 )6 is diamagnetic and symmetric, with all ligands magnetically equivalent. Quantum computations cross-validate NMR results and reveal degenerate delocalized HOMO orbitals, indicating aromaticity. Ligand substitution keeps the inorganic core nearly intact. After losing one electron, the unpaired electron in [Co6 Se8 (PEt3 )6 ]+1 is delocalized, causing paramagnetism and a delocalized electron spin. Notably, this feature of electron/spin delocalization over a large cluster is attractive for special single-electron devices.

Phe-Gly motifs drive fibrillization of TDP-43's prion-like domain condensates.

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.

Rotating Frame Relaxation in Magic Angle Spinning Solid State NMR, a Promising Tool for Characterizing Biopolymer Motion.

Magic angle spinning NMR rotating frame relaxation measurements provide a unique experimental window into biomolecules dynamics, as is illustrated by numerous recent applications. We discuss experimental strategies for this class of experiments, with a particular focus on systems where motion-driven modulation of the chemical shift interaction is the main mechanism for relaxation. We also explore and describe common strategies for interpreting the data sets to extract motion time scale, activation energy, and angle or order parameters from rotating frame relaxation data. Using model free analysis and numerical simulations, including time domain treatment, we explore conditions under which it is possible to obtain accurate and precise information about the time scales of motions. Overall, with rapid technical advances in solid state NMR, there is a bright future for this class of studies.

Probing allosteric coupling in a constitutively open mutant of the ion channel KcsA using solid-state NMR.

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation.

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a "prion-related domain" or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.

Iterative Synthesis of Contorted Macromolecular Ladders for Fast-Charging and Long-Life Lithium Batteries.

We report here an iterative synthesis of long helical perylene diimide (hPDI[n]) nanoribbons with a length up to 16 fused benzene rings. These contorted, ladder-type conjugated, and atomically precise nanoribbons show great potential as organic fast-charging and long-lifetime battery cathodes. By tuning the length of the hPDI[n] oligomers, we can simultaneously modulate the electrical conductivity and ionic diffusivity of the material. The length of the ladders adjusts both the conjugation for electron transport and the contortion for lithium-ion transport. The longest oligomer, hPDI[6], when fabricated as the cathode in lithium batteries, features both high electrical conductivity and high ionic diffusivity. This electrode material exhibits a high power density and can be charged in less than 1 min to 66% of its maximum capacity. Remarkably, this material also has exceptional cycling stability and can operate for up to 10,000 charging-discharging cycles without any appreciable capacity decay. The design principles described here chart a clear path for organic battery electrodes that are sustainable, fast-charging, and long lasting.

Identifying coupled clusters of allostery participants through chemical shift perturbations.

Allosteric couplings underlie many cellular signaling processes and provide an exciting avenue for development of new diagnostics and therapeutics. A general method for identifying important residues in allosteric mechanisms would be very useful, but remains elusive due to the complexity of long-range phenomena. Here, we introduce an NMR method to identify residues involved in allosteric coupling between two ligand-binding sites in a protein, which we call chemical shift detection of allostery participants (CAP). Networks of functional groups responding to each ligand are defined through correlated NMR perturbations. In this process, we also identify allostery participants, groups that respond to both binding events and likely play a role in the coupling between the binding sites. Such residues exhibit multiple functional states with distinct NMR chemical shifts, depending on binding status at both binding sites. Such a strategy was applied to the prototypical ion channel KcsA. We had previously shown that the potassium affinity at the extracellular selectivity filter is strongly dependent on proton binding at the intracellular pH sensor. Here, we analyzed proton and potassium binding networks and identified groups that depend on both proton and potassium binding (allostery participants). These groups are viewed as candidates for transmitting information between functional units. The vital role of one such identified amino acid was validated through site-specific mutagenesis, electrophysiology functional studies, and NMR-detected thermodynamic analysis of allosteric coupling. This strategy for identifying allostery participants is likely to have applications for many other systems.

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel.

As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

Scaled recoupling of chemical shift anisotropies at high magnetic fields under MAS with interspersed C-elements.

The power of chemical shift anisotropy (CSA) measurements for probing structure and dynamics of molecules has been long recognized. NMR pulse sequences that allow measurement of CSA values in an indirect dimension of a protein correlation spectrum have been employed for aliphatic groups, but for practical reasons, carbonyl functional groups have been little studied, despite the fact that carbonyls are expected to give particularly varied and informative CSA values. Specifically, the wide spectral widths of carbonyl tensors make their measurements difficult with typically attainable spectrometer settings. We present here an extended family of experiments that enable the recovery of static CSA lineshapes in an indirect dimension of magic angle spinning (MAS) solid-state NMR experiments, except for various real valued scaling factors. The experiment is suitable for uniformly labeled material, at moderate MAS rates (10 kHz-30 kHz) and at higher magnetic fields (ν0H > 600 MHz). Specifically, the experiments are based on pulse sequence elements from a previous commonly used pulse sequence for CSA measurement, recoupling of chemical shift anisotropy (ROCSA), while modification of scaling factors is achieved by interspersing different blocks of C-elements of the same Cnn 1 cycle. Using experimental conditions similar to the parent ROCSA sequence, a CSA scaling factor between 0 and 0.272 can be obtained, thus allowing a useful practical range of possibilities in experimental conditions for measurement of larger CSA values. Using these blocks, it is also possible to make a constant-time CSA recoupling sequence. The effectiveness of this approach, fROCSA, is shown on model compounds 1-13C-Gly, U-13C,15N-l-His, and microcrystalline U-13C,15N-Ubiquitin.

Transmembrane allosteric energetics characterization for strong coupling between proton and potassium ion binding in the KcsA channel.

The slow spontaneous inactivation of potassium channels exhibits classic signatures of transmembrane allostery. A variety of data support a model in which the loss of K+ ions from the selectivity filter is a major factor in promoting inactivation, which defeats transmission, and is allosterically coupled to protonation of key channel activation residues, more than 30 Å from the K+ ion binding site. We show that proton binding at the intracellular pH sensor perturbs the potassium affinity at the extracellular selectivity filter by more than three orders of magnitude for the full-length wild-type KcsA, a pH-gated bacterial channel, in membrane bilayers. Studies of F103 in the hinge of the inner helix suggest an important role for its bulky sidechain in the allosteric mechanism; we show that the energetic strength of coupling of the gates is strongly altered when this residue is mutated to alanine. These results provide quantitative site-specific measurements of allostery in a bilayer environment, and highlight the power of describing ion channel gating through the lens of allosteric coupling.

Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization.

An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR.

Magic angle spinning solid state NMR studies of biological macromolecules [1-3] have enabled exciting studies of membrane proteins [4,5], amyloid fibrils [6], viruses, and large macromolecular assemblies [7]. Dynamic nuclear polarization (DNP) provides a means to enhance detection sensitivity for NMR, particularly for solid state NMR, with many recent biological applications and considerable contemporary efforts towards elaboration and optimization of the DNP experiment. This review explores precedents and innovations in biological DNP experiments, especially highlighting novel chemical biology approaches to introduce the radicals that serve as a source of polarization in DNP experiments.

Store-directed price promotions and communications strategies improve healthier food supply and demand: impact results from a randomized controlled, Baltimore City store-intervention trial.

OBJECTIVE: Small food store interventions show promise to increase healthy food access in under-resourced areas. However, none have tested the impact of price discounts on healthy food supply and demand. We tested the impact of store-directed price discounts and communications strategies, separately and combined, on the stocking, sales and prices of healthier foods and on storeowner psychosocial factors. DESIGN: Factorial design randomized controlled trial. SETTING: Twenty-four corner stores in low-income neighbourhoods of Baltimore City, MD, USA. SUBJECTS: Stores were randomized to pricing intervention, communications intervention, combined pricing and communications intervention, or control. Stores that received the pricing intervention were given a 10-30 % price discount by wholesalers on selected healthier food items during the 6-month trial. Communications stores received visual and interactive materials to promote healthy items, including signage, taste tests and refrigerators. RESULTS: All interventions showed significantly increased stock of promoted foods v. CONTROL: There was a significant treatment effect for daily unit sales of healthy snacks (ß=6·4, 95 % CI 0·9, 11·9) and prices of healthy staple foods (ß=-0·49, 95 % CI -0·90, -0·03) for the combined group v. control, but not for other intervention groups. There were no significant intervention effects on storeowner psychosocial factors. CONCLUSIONS: All interventions led to increased stock of healthier foods. The combined intervention was effective in increasing sales of healthier snacks, even though discounts on snacks were not passed to the consumer. Experimental research in small stores is needed to understand the mechanisms by which store-directed price promotions can increase healthy food supply and demand.

Transmembrane allosteric coupling of the gates in a potassium channel.

It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98-103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.

Microtia and Social Media: Patient Versus Physician Perspective of Quality of Information.

INTRODUCTION: Previous research demonstrates that patients seek high-quality information on the World Wide Web, especially in rare conditions such as microtia. Social media has overtaken other sources of patient information but quality remains untested. This study quantifies the quality of information for patients with Microtia on social media compared with nonsocial media websites and compares physician and patient scoring on quality using the DISCERN tool. METHODS: In phase 1, quality of the top 100 websites featuring information "Microtia" was ranked according to quality score and position on Google showing the position of social media websites among other nonsocial media websites. Phase 2 involved independent scoring of websites on microtia compared with a patient group with microtia to test whether physicians score differently to patients with t test comparison. RESULTS: Social media websites account for 2% of the scored websites with health providers linking to social media. Social media websites were among the highest ranked on Google. No correlation was found between the quality of information and Google rank. Social media scored higher than nonsocial media websites regarding quality of information on microtia. No significant difference existed between physician and patient quality of information scores on social media and nonsocial media websites (p 1.033). CONCLUSION: Physicians and patients objectively score microtia websites alike. Social media websites have higher use despite being few in number compared with nonsocial media websites. Physicians providing links to social media on information websites on rare conditions such as microtia are engaging in current information-seeking trends.

Multidimensional Solid-State Nuclear Magnetic Resonance of a Functional Multiprotein Chemoreceptor Array.

The bacterial chemoreceptor complex governs signal detection and the upstream elements of chemotactic behavior, but the detailed molecular mechanism is still unclear. We have assembled nativelike functional arrays of an aspartate receptor cytoplasmic fragment (CF) with its two cytoplasmic protein partners (CheA and CheW) for solid-state nuclear magnetic resonance (NMR) studies of structural changes involved in signaling. In this initial study of the uniformly (13)C- and (15)N-enriched CF in these >13.8 MDa size arrays, residue-type assignments are made for amino acids that together make up 90% of the protein. We demonstrate that homo- and heteronuclear two-dimensional spectra are consistent with structure-based chemical shift predictions: a number of major assignable correlations are consistent with the predominantly α-helical secondary structure, and minor correlations are consistent with the disordered C-terminal tail. Sub-parts per million line widths and spectral changes upon freezing of samples suggest these arrays are structurally homogeneous and sufficiently immobilized for efficient solid-state NMR.

Cardiolipin interaction with subunit c of ATP synthase: solid-state NMR characterization.

The interaction of lipids with subunit c from F1F0 ATP synthase is studied by biophysical methods. Subunit c from both Escherichia coli and Streptococcus pneumoniae interacts and copurifies with cardiolipin. Solid state NMR data on oligomeric rings of F0 show that the cardiolipin interacts with the c subunit in membrane bilayers. These studies offer strong support for the hypothesis that F0 has specific interactions with cardiolipin.