Beta-Cardiotoxin: A novel protein isolated from the venom of Ophiophagus hannah (king cobra) showing beta-blocker activity

Ph.DDOCTOR OF PHILOSOPH

A Two-Step Method for Obtaining Highly Pure Cas9 Nuclease for Genome Editing, Biophysical, and Structural Studies

Cas9 is a site-specific RNA-guided endonuclease (RGEN) that can be used for precise genome editing in various cell types from multiple species. Ribonucleoprotein (RNP) complexes, which contains the Cas9 protein in complex with a guide RNA, are sufficient for the precise editing of genomes in various cells. This DNA-free method is more specific in editing the target sites and there is no integration of foreign DNA into the genome. Also, there are ongoing studies into the interactions of Cas9 protein with modified guide RNAs, as well as structure-activity studies of Cas9 protein and its variants. All these investigations require highly pure Cas9 protein. A single-step metal affinity enrichment yielding impure Cas9 is the most common method of purification described. This is sufficient for many gene editing applications of this protein. However, to obtain Cas9 of higher purity, which might be essential for biophysical characterization, chemical modifications, and structural investigations, laborious multi-step protocols are employed. Here, we describe a two-step Cas9 purification protocol that uses metal affinity enrichment followed by cation exchange chromatography. This simple method can yield a milligram of highly pure Cas9 protein per liter of culture in a single day

Molecular mechanisms in the selective basal activation of pyrabactin receptor 1: Comparative analysis of mutants

Pyrabactin receptors (PYR) play a central role in abscisic acid (ABA) signal transduction; they are ABA receptors that inhibit type 2C protein phosphatases (PP2C). Molecular aspects contributing to increased basal activity of PYR against PP2C are studied by molecular dynamics (MD) simulations. An extensive series of MD simulations of the apo-form of mutagenized PYR1 as a homodimer and in complex with homology to ABA-insensitive 1 (HAB1) phosphatase are reported. In order to investigate the detailed molecular mechanisms mediating PYR1 activity, the MD data was analyzed by essential collective dynamics (ECD), a novel approach that allows the identification, with atomic resolution, of persistent dynamic correlations based on relatively short MD trajectories. Employing the ECD method, the effects of select mutations on the structure and dynamics of the PYR1 complexes were investigated and considered in the context of experimentally determined constitutive activities against HAB1. Approaches to rationally design constitutively active PYR1 constructs to increase PP2C inhibition are discussed.Peer reviewed: YesNRC publication: Ye

Metabolomics and Cheminformatics Analysis of Antifungal Function of Plant Metabolites

Fusarium head blight (FHB), primarily caused by Fusarium graminearum, is a devastating disease of wheat. Partial resistance to FHB of several wheat cultivars includes specific metabolic responses to inoculation. Previously published studies have determined major metabolic changes induced by pathogens in resistant and susceptible plants. Functionality of the majority of these metabolites in resistance remains unknown. In this work we have made a compilation of all metabolites determined as selectively accumulated following FHB inoculation in resistant plants. Characteristics, as well as possible functions and targets of these metabolites, are investigated using cheminformatics approaches with focus on the likelihood of these metabolites acting as drug-like molecules against fungal pathogens. Results of computational analyses of binding properties of several representative metabolites to homology models of fungal proteins are presented. Theoretical analysis highlights the possibility for strong inhibitory activity of several metabolites against some major proteins in Fusarium graminearum, such as carbonic anhydrases and cytochrome P450s. Activity of several of these compounds has been experimentally confirmed in fungal growth inhibition assays. Analysis of anti-fungal properties of plant metabolites can lead to the development of more resistant wheat varieties while showing novel application of cheminformatics approaches in the analysis of plant/pathogen interactions

Probing allocrite preferences of two naturally occurring variants of the wheat LR34 ABC transporter

For almost a century, the wheat Lr34 gene has conferred durable resistance against fungal rust diseases. While sequence homology predicts a putative ATP binding cassette transporter, the molecules that are transported (allocrites) by the encoded LR34 variants, and any associated mechanism of resistance, remain enigmatic. Here, the in vitro transport characteristics of two naturally occurring Lr34 variants (that differ in their ability to mediate disease resistance; Lr34sus and Lr34res) are investigated. Initially a method to express and purify recombinant LR34Sus and LR34Res pseudo half-molecules from Saccharomyces cerevisiae, is described. Subsequently a semi-targeted chlorophyll catabolite (CC) extraction from Lr34res-expressing wheat plants was performed based on previous reports highlighting increased accumulation of CCs in Lr34res-expressing flag leaves. Following partial biochemical characterization, this extract was applied to an LR34 in vitro proteoliposome transport assay. Mass spectroscopic analyses of transported metabolites revealed that LR34Sus imported a wheat metabolite of 618 Da and that the LR34Res transporter did not. While the identity of the LR34Sus transported metabolite remains to be confirmed and any allocrites of LR34Res remain to be detected, this work demonstrates that these variants have different allocrite preferences, a finding that may be relevant to the mechanism of disease resistance.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

THERAPEUTIC USES OF BETA-ANTAGONISTS

US20100062980A1Published Applicatio

Snapshot of proteomic changes in Aspergillus oryzae during various stages of fermentative processing of pea protein isolate

Pea (Pisum sativum) is one of the most abundant and sustainable alternate source of protein. Although pea proteins have good quantities of most of the essential amino acids, they have a limited supply of tryptophan, methionine and cysteine. Moreover, pea proteins have poor techno-functional properties compared to proteins from animal sources, limiting their use in certain food applications. Bioprocessing techniques like solid-state fermentation (SSF) and enzymatic processing have been explored to improve the nutrient profile and functionality of pea proteins. However, there is a lack of information about proteomic changes in the food matrix during fermentation of the pea substrate. In this research, samples during SSF of pea protein isolate with Aspergillus oryzae were used for shotgun mass spectrometry (LC-MS/MS) analysis to identify the underlying functional pathways which play direct or indirect roles in enabling the colonization of the substrate leading to potential improvement of functional and nutritional value of pea protein. Results revealed the identity of A. oryzae proteins involved in different metabolic pathways that differed during various stages of SSF. Among them, methionine synthase was identified as an abundant protein, which catalyzes methionine biosynthesis. This might suggest how fermentation processes could be used to improve the presence of sulfur containing amino acids to rebalance the essential amino acid profile and improve the nutritional quality of pea proteins

Abscisic acid binding by the PYR1 ligand pocket induces gate-latch-locking.

<p>(A) – Structure of the apo-PYR1, gate open [PDB ID 3K3K, chain A]. (B) – Structure of ABA-bound PYR1, gate closed [PDB ID 3K3K, chain B]. The lock mechanism involves both direct and water/ions-mediated interactions of residues from gate (residues 85–89) and latch (residues 115–117), as well as hydrophobic interactions and hydrogen bonds throughout the binding pocket's surface. Residues which contribute to hydrogen binding in gate and latch are labeled and shown by orange sticks, while hydrophobic residues in the neighborhood of ABA (colored yellow) are shown by purple sticks. The allosteric rearrangement of gate and latch loops forms a surface for successful PP2C binding. Upon the binding, a conserved PP2C tryptophan 385 (not shown) is inserted between gate and latch and forms water-mediated hydrogen bond with ABA <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi.1003114-Nishimura1" target="_blank">[9]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi.1003114-Melcher1" target="_blank">[11]</a>.</p

Normalized main-chain flexibility profiles of PYR1 monomers

<p> (solid lines) over-imposed on B-factors of the corresponding starting crystallographic structures (chains A and B from PDB entry 3K3K <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi.1003114-Nishimura1" target="_blank">[9]</a>, dashed lines). In the plot, red color represents PYR1 in ABA-bound, closed-lid conformation and blue color represents ABA-free, open lid conformation.</p