Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SulfETF), tryptophan synthases from Salmonella typhimurium (StTS) and the dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

TapA acts as specific chaperone in TasA filament formation by strand complementation.

Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell-cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of ß-sandwich subunits. The secondary structure around the intercalated N-terminal strand ß0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface.

A Membrane-Permeable and Immobilized Metal Affinity Chromatography (IMAC) Enrichable Cross-Linking Reagent to Advance In†Vivo Cross-Linking Mass Spectrometry.

Cross-linking mass spectrometry (XL-MS) is an attractive method for the proteome-wide characterization of protein structures and interactions. Currently, the depth of in vivo XL-MS studies is lagging behind the established applications to cell lysates, because cross-linking reagents that can penetrate intact cells and strategies to enrich cross-linked peptides lack efficiency. To tackle these limitations, we have developed a phosphonate-containing cross-linker, tBu-PhoX, that efficiently permeates various biological membranes and can be robustly enriched using routine immobilized metal ion affinity chromatography. We have established a tBu-PhoX-based in vivo XL-MS approach that enables cross-links in intact human cells to be identified in high numbers with substantially reduced analysis time. Collectively, the developed cross-linker and XL-MS approach pave the way for the comprehensive XL-MS characterization of living systems.

NMR structure and dynamics of Q4DY78, a conserved kinetoplasid-specific protein from Trypanosoma cruzi.

The 106-residue protein Q4DY78 (UniProt accession number) from Trypanosoma cruzi is highly conserved in the related kinetoplastid pathogens Trypanosoma brucei and Leishmania major. Given the essentiality of its orthologue in T. brucei, the high sequence conservation with other trypanosomatid proteins, and the low sequence similarity with mammalian proteins, Q4DY78 is an attractive protein for structural characterization. Here, we solved the structure of Q4DY78 by solution NMR and evaluated its backbone dynamics. Q4DY78 is composed of five α -helices and a small, two-stranded antiparallel ß-sheet. The backbone RMSD is 0.22 ± 0.05 Å for the representative ensemble of the 20 lowest-energy structures. Q4DY78 is overall rigid, except for N-terminal residues (V8 to I10), residues at loop 4 (K57 to G65) and residues at the C-terminus (F89 to F112). Q4DY78 has a short motif FPCAP that could potentially mediate interactions with the host cytoskeleton via interaction with EVH1 (Drosophila Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) homology 1) domains. Albeit Q4DY78 lacks calcium-binding motifs, its fold resembles that of eukaryotic calcium-binding proteins such as calcitracin, calmodulin, and polcacin Bet V4. We characterized this novel protein with a calcium binding fold without the capacity to bind calcium.

Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling.

Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ-Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure-activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50∼50 µM) and Western blotting of ß-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (KD=2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.

Crystal structure of Q4D6Q6, a conserved kinetoplastid-specific protein from Trypanosoma cruzi.

Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ßßαßßαßß topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction.

pH-Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes.

Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen-evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn4 CaO5 cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pKa values of water-accessible aspartate and glutamate side-chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pKa values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH-dependent changes in backbone chemical shifts in the area of the lumen-exposed loops are observed, indicating conformational changes. In conclusion, we present a site-specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis.

The Association Between Parental Depression and Child Psychosocial Intervention Outcomes: Directions for Future Research.

Recent studies suggest that parental depressive symptoms may affect a child's ability to benefit from interventions for anxiety and depression. This article reviews the current literature, suggesting that, when parents experience current depressive symptoms, children are less likely to benefit from psychosocial interventions for anxiety and depression. Opportunities for future research are discussed, including moderators and mechanisms of the association between parental depressive symptoms and child intervention outcomes.

The companion of cellulose synthase 1 confers salt tolerance through a Tau-like mechanism in plants.

Microtubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). Here we outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed points through four conserved hydrophobic regions. By NMR and live cell analyses we reveal that two neighboring residues in the first hydrophobic binding motif are crucial for the microtubule interaction. The microtubule-binding mechanism of CC1 is reminiscent to that of the prominent neuropathology-related protein Tau, indicating evolutionary convergence of MAP functions across animal and plant cells.

The protofilament architecture of a de novo designed coiled coil-based amyloidogenic peptide.

Amyloid fibrils are polymers formed by proteins under specific conditions and in many cases they are related to pathogenesis, such as Parkinson's and Alzheimer's diseases. Their hallmark is the presence of a ß-sheet structure. High resolution structural data on these systems as well as information gathered from multiple complementary analytical techniques is needed, from both a fundamental and a pharmaceutical perspective. Here, a previously reported de novo designed, pH-switchable coiled coil-based peptide that undergoes structural transitions resulting in fibril formation under physiological conditions has been exhaustively characterized by transmission electron microscopy (TEM), cryo-TEM, atomic force microscopy (AFM), wide-angle X-ray scattering (WAXS) and solid-state NMR (ssNMR). Overall, a unique 2-dimensional carpet-like assembly composed of large coexisiting ribbon-like, tubular and funnel-like structures with a clearly resolved protofilament substructure is observed. Whereas electron microscopy and scattering data point somewhat more to a hairpin model of ß-fibrils, ssNMR data obtained from samples with selectively labelled peptides are in agreement with both, hairpin structures and linear arrangements.

Structural changes of TasA in biofilm formation of Bacillus subtilis.

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, ß-sheet-rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level.

Temperature dependence of cross-effect dynamic nuclear polarization in rotating solids: advantages of elevated temperatures.

Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K. The combined effects of enhancement, depolarization, and proton longitudinal relaxation are surprisingly sample-specific. At 200 K, DNP on SH3 domain standard samples yields a 15-fold increase in signal-to-noise over a sample without radicals. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours, respectively. Decreasing enhancements with increasing 2H-content at the CH2 sites of the TEMPO rings in CD3-TOTAPOL highlight the importance of protons in a sphere of 4-6 Å around the nitroxyl group, presumably for polarization pickup from electron spins.

Chemical shift assignments and secondary structure prediction for Q4DY78, a conserved kinetoplastid-specific protein from Trypanosoma cruzi.

Trypanosoma cruzi, Trypanosma brucei and Leishmania spp. are kinetoplastid protozoa causative agents of Chagas disease, sleeping sickness and leishmaniasis, respectively, neglected tropical diseases estimated to infect millions of people worldwide. Their genome sequencing has revealed approximately 50 % of genes encoding hypothetical proteins of unknown function, opening possibilities for novel target identification and drug discovery. Q4DY78 is a putative essential protein from T. cruzi conserved in the related kinetoplastids and divergent from mammalian host proteins. Here we report the (1)H, (15)N, and (13)C chemical shift assignments and secondary structure analysis of the Q4DY78 protein as basis for NMR structure determination, functional analysis and drug screening.

Increasing Understanding in Children of Depressed Parents: Predictors and Moderators of Intervention Response.

We evaluated predictors and moderators of differential response to two family-based depression prevention programs for families with a depressed parent: a clinician-facilitated intervention and a lecture group intervention. Individual and family level variables were examined using regression analyses with generalized estimating equations. For the outcome of child understanding of depression, parental changes in child-related behaviors and attitudes predicted greater child understanding (p < 0.001). For the parent outcome of behavior and attitude change, across intervention conditions, younger parent age (p < 0.05), female parent gender (p < 0.01), more chronic and severe parental depression history (p < 0.05), lower SES (p < 0.05), and single-parent status (p < 0.05) were associated with better outcomes across conditions. Effect sizes were moderate, ranging from 0.4 to 0.7 SD. Family and marital functioning were not found to be predictors of any outcomes. When both parents were depressed at baseline, there was no difference in the clinician- versus lecture-based approach, and when only the father was depressed, families reported more changes with the clinician condition than with the lecture condition (p < 0.05). Findings from this study can help identify intervention strategies that are appropriate for different types of at-risk individuals and families.

Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-trans and 13-cis Retinal Isomers.

Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13)═C(14) and C(15)═N bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13)═C(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.

An internet-based adolescent depression preventive intervention: study protocol for a randomized control trial.

BACKGROUND: The high prevalence of major depressive disorder in adolescents and the low rate of successful treatment highlight a pressing need for accessible, affordable adolescent depression prevention programs. The Internet offers opportunities to provide adolescents with high quality, evidence-based programs without burdening or creating new care delivery systems. Internet-based interventions hold promise, but further research is needed to explore the efficacy of these approaches and ways of integrating emerging technologies for behavioral health into the primary care system. METHODS/DESIGN: We developed a primary care Internet-based depression prevention intervention, Competent Adulthood Transition with Cognitive Behavioral Humanistic and Interpersonal Training (CATCH-IT), to evaluate a self-guided, online approach to depression prevention and are conducting a randomized clinical trial comparing CATCH-IT to a general health education Internet intervention. This article documents the research framework and randomized clinical trial design used to evaluate CATCH-IT for adolescents, in order to inform future work in Internet-based adolescent prevention programs. The rationale for this trial is introduced, the current status of the study is reviewed, and potential implications and future directions are discussed. DISCUSSION: The current protocol represents the only current, systematic approach to connecting at-risk youth with self-directed depression prevention programs in a medical setting. This trial undertakes the complex public health task of identifying at-risk individuals through mass screening of the general primary care population, rather than solely relying on volunteers recruited over the Internet, and the trial design provides measures of both symptomatic and diagnostic clinical outcomes. At the present time, we have enrolled N = 234 adolescents/expected 400 and N = 186 parents/expected 400 in this trial, from N = 6 major health systems. The protocol described here provides a model for a new generation of interventions that blend substantial computer-based instruction with human contact to intervene to prevent mental disorders such as depression. Because of the potential for broad generalizability of this model, the results of this study are important, as they will help develop the guidelines for preventive interventions with youth at-risk for the development of depressive and other mental disorders. CLINICAL TRIAL REGISTRY: NCT01893749 date 6 May 2012.

Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning.

The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

Development of a technology-based behavioral vaccine to prevent adolescent depression: A health system integration model.

Efforts to prevent depression have become a key health system priority. Currently, there is a high prevalence of depression among adolescents, and treatment has become costly due to the recurrence patterns of the illness, impairment among patients, and the complex factors needed for a treatment to be effective. Primary care may be the optimal location to identify those at risk by offering an Internet-based preventive intervention to reduce costs and improve outcomes. Few practical interventions have been developed. The models for Internet intervention development that have been put forward focus primarily on the Internet component rather than how the program fits within a broader context. This paper describes the conceptualization for developing technology based preventive models for primary care by integrating the components within a behavioral vaccine framework. CATCH-IT (Competent Adulthood Transition with Cognitive-behavioral, Humanistic and Interpersonal Training) has been developed and successfully implemented within various health systems over a period of 14 years among adolescents and young adults aged 13-24.

AhR sensing of bacterial pigments regulates antibacterial defence.

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.