Concepts and considerations for enhancing RNAi efficiency in phytopathogenic fungi for RNAi-based crop protection using nanocarrier-mediated dsRNA delivery systems.

Existing, emerging, and reemerging strains of phytopathogenic fungi pose a significant threat to agricultural productivity globally. This risk is further exacerbated by the lack of resistance source(s) in plants or a breakdown of resistance by pathogens through co-evolution. In recent years, attenuation of essential pathogen gene(s) double-stranded (ds) RNA-mediated RNA interference (RNAi) in host plants, a phenomenon known as host-induced gene silencing, has gained significant attention as a way to combat pathogen attack.

Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.

CLEC16A is an E3 ubiquitin ligase that regulates mitochondrial quality control through mitophagy and is associated with over 20 human diseases. CLEC16A forms a complex with another E3 ligase, RNF41, and a ubiquitin-specific peptidase, USP8; however, regions that regulate CLEC16A activity or the assembly of the tripartite mitophagy regulatory complex are unknown. Here, we report that CLEC16A contains an internal intrinsically disordered protein region (IDPR) that is crucial for CLEC16A function and turnover.

An intrinsically disordered protein region encoded by the human disease gene regulates mitophagy.

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. However, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control.

Distinct conformational dynamics and allosteric networks in alpha tryptophan synthase during active catalysis.

Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR.

Different Solvent and Conformational Entropy Contributions to the Allosteric Activation and Inhibition Mechanisms of Yeast Chorismate Mutase.

Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector.

Diastereoselective multi-component tandem condensation: synthesis of 2-amino-4-(2-furanone)-4H-chromene-3-carbonitriles.

A general strategy for a one-pot stereoselective synthesis of 2-amino-4-(2-furanone)-4H-chromene-3-carbonitriles by reaction of salicylaldehyde, malononitrile and butenolides via a tandem Knoevenagel/Pinner/vinylogous Michael condensation is presented. The β,γ-butenolides gave a syn-selective MCR adduct with a dr up to 11.5 : 1.

Coordinated Network Changes across the Catalytic Cycle of Alpha Tryptophan Synthase.

Networks of noncovalent interactions are important for protein structural dynamics. We used nuclear magnetic resonance chemical shift covariance analyses on an inactive variant of the alpha subunit of tryptophan synthase to map amino acid interaction networks across its catalytic cycle. Although some network connections were common to every enzyme state, many of the network connections strengthened or weakened over the catalytic cycle; these changes were highly coordinated.

Solution Ensemble of the C-Terminal Domain from the Transcription Factor Pdx1 Resembles an Excluded Volume Polymer.

The pancreatic and duodenal homeobox 1 (Pdx1) is an essential pancreatic transcription factor. The C-terminal intrinsically disordered domain of Pdx1 (Pdx1-C) has a heavily biased amino acid composition; most notably, 18 of 83 residues are proline, including a hexaproline cluster near the middle of the chain. For these reasons, Pdx1-C is an attractive target for structure characterization, given the availability of suitable methods.

Millisecond Timescale Motions Connect Amino Acid Interaction Networks in Alpha Tryptophan Synthase.

Tryptophan synthase is a model system for understanding allosteric regulation within enzyme complexes. Amino acid interaction networks were previously delineated in the isolated alpha subunit (αTS) in the absence of the beta subunit (βTS). The amino acid interaction networks were different between the ligand-free enzyme and the enzyme actively catalyzing turnover.

Assigning methyl resonances for protein solution-state NMR studies.

Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has substantially expanded the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to grow, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (>50 kDa) proteins.

Discrete-State Kinetics Model for NMR-Based Analysis of Protein Translocation on DNA at Equilibrium.

In the target DNA search process, sequence-specific DNA-binding proteins first nonspecifically bind to DNA and stochastically move from one site to another before reaching their targets. To rigorously assess how the translocation process influences NMR signals from proteins interacting with nonspecific DNA, we incorporated a discrete-state kinetic model for protein translocation on DNA into the McConnell equation. Using this equation, we simulated line shapes of NMR signals from proteins undergoing translocations on DNA through sliding, dissociation/reassociation, and intersegment transfer.

Engineering double-stranded RNA binding activity into the Drosha double-stranded RNA binding domain results in a loss of microRNA processing function.

Canonical processing of miRNA begins in the nucleus with the Microprocessor complex, which is minimally composed of the RNase III enzyme Drosha and two copies of its cofactor protein DGCR8. In structural analogy to most RNase III enzymes, Drosha possesses a modular domain with the double-stranded RNA binding domain (dsRBD) fold. Unlike the dsRBDs found in most members of the RNase III family, the Drosha-dsRBD does not display double-stranded RNA binding activity; perhaps related to this, the Drosha-dsRBD amino acid sequence does not conform well to the canonical patterns expected for a dsRBD.

Assessing Coupled Protein Folding and Binding Through Temperature-Dependent Isothermal Titration Calorimetry.

Broad interest in the thermodynamic driving forces of coupled macromolecular folding and binding is motivated by the prevalence of disorder-to-order transitions observed when intrinsically disordered proteins (IDPs) bind to their partners. Isothermal titration calorimetry (ITC) is one of the few methods available for completely evaluating the thermodynamic parameters describing a protein-ligand binding event. Significantly, when the effective ΔH° for the coupled folding and binding process is determined by ITC in a temperature series, the constant-pressure heat capacity change (ΔCp) associated with these coupled equilibria is experimentally accessible, offering a unique opportunity to investigate the driving forces behind them.

Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods.

There is an extraordinary need to describe the structures of intrinsically disordered proteins (IDPs) due to their role in various biological processes involved in signaling and transcription. However, general study of IDPs by NMR spectroscopy is limited by the poor (1)H amide chemical shift dispersion typically observed in their spectra. Recently, (13)C direct-detected NMR spectroscopy has been recognized as enabling broad structural study of IDPs.

Real-time kinetics of high-mobility group box 1 (HMGB1) oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.

Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation.

Ensemble analysis of primary microRNA structure reveals an extensive capacity to deform near the Drosha cleavage site.

Most noncoding RNAs function properly only when folded into complex three-dimensional (3D) structures, but the experimental determination of these structures remains challenging. Understanding of primary microRNA (miRNA) maturation is currently limited by a lack of determined structures for nonprocessed forms of the RNA. SHAPE chemistry efficiently determines RNA secondary structural information with single-nucleotide resolution, providing constraints suitable for input into MC-Pipeline for refinement of 3D structure models.

The role of human Dicer-dsRBD in processing small regulatory RNAs.

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC).

Asymmetrical roles of zinc fingers in dynamic DNA-scanning process by the inducible transcription factor Egr-1.

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNA-scanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA.

NMR studies of translocation of the Zif268 protein between its target DNA Sites.

Zif268 is a zinc-finger protein containing three Cys(2)-His(2)-type zinc-finger domains that bind the target DNA sequence GCGTGGGCG in a cooperative manner. In this work, we characterized translocation of the Zif268 protein between its target DNA sites using NMR spectroscopy. The residual dipolar coupling data and NMR chemical shift data suggested that the structure of the sequence-specific complex between Zif268 and its target DNA in solution is the same as the crystal structure.

Redox properties of the A-domain of the HMGB1 protein.

The High Mobility Group B1 (HMGB1) protein plays important roles in both intracellular (reductive) and extracellular (oxidative) environments. We have carried out quantitative investigations of the redox chemistry involving Cys22 and Cys44 of the HMGB1 A-domain, which form an intramolecular disulfide bond. Using NMR spectroscopy, we analyzed the real-time kinetics of the redox reactions for the A-domain in glutathione and thioredoxin systems, and also determined the standard redox potential.

Observing in-phase single-quantum 15N multiplets for NH2/NH3+ groups with two-dimensional heteronuclear correlation spectroscopy.

Two-dimensional (2D) F1-(1)H-coupled HSQC experiments provide 3:1:1:3 and 1:0:1 multiplets for AX(3) and AX(2) spin systems, respectively. These multiplets occur because, in addition to the 2S(y)H(z)(a)-->2S(y)H(z)(a) process, the coherence transfers such as 2S(y)H(z)(a)-->2S(y)H(z)(b) occurring in t(1) period provide detectable magnetization during the t(2) period. Here, we present a 2D F1-(1)H-coupled (1)H-(15)N heteronuclear correlation experiment that provides a 1:3:3:1 quartet for AX(3) spin system and a 1:2:1 triplet for AX(2).

TROSY-based z-exchange spectroscopy: application to the determination of the activation energy for intermolecular protein translocation between specific sites on different DNA molecules.

A two-dimensional TROSY-based z-exchange 1H-15N correlation experiment for the quantitative analysis of kinetic processes in the slow exchange regime is presented. The pulse scheme converts the product operator terms Nz into 2NzHz and 2NzHz into -Nz in the middle of the z-mixing period, thereby suppressing the buildup of spurious semi-TROSY peaks arising from the different relaxation rates for the Nz and 2NzHz terms and simplifying the behavior of longitudinal magnetization for an exchanging system during the mixing period. Theoretical considerations and experimental data demonstrate that the TROSY-based z-exchange experiment permits quantitative determination of rate constants using the same procedure as that for the conventional non-TROSY 15Nz-exchange experiment.

Bhageerath: an energy based web enabled computer software suite for limiting the search space of tertiary structures of small globular proteins.

We describe here an energy based computer software suite for narrowing down the search space of tertiary structures of small globular proteins. The protocol comprises eight different computational modules that form an automated pipeline. It combines physics based potentials with biophysical filters to arrive at 10 plausible candidate structures starting from sequence and secondary structure information.