Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening.

Target-based discovery of first-in-class therapeutics demands an in-depth understanding of the molecular mechanisms underlying human diseases. Precise measurements of cellular and biochemical activities are critical to gain mechanistic knowledge of biomolecules and their altered function in disease conditions. Such measurements enable the development of intervention strategies for preventing or treating diseases by modulation of desired molecular processes.

Mutational scan inferred binding energetics and structure in intrinsically disordered protein CcdA.

Unlike globular proteins, mutational effects on the function of Intrinsically Disordered Proteins (IDPs) are not well-studied. Deep Mutational Scanning of a yeast surface displayed mutant library yields insights into sequence-function relationships in the CcdA IDP. The approach enables facile prediction of interface residues and local structural signatures of the bound conformation.

Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region.

The genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding.

Mechanistic insights into global suppressors of protein folding defects.

Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution.

Structural and functional determinants inferred from deep mutational scans.

Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase.

Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries.

Although there have been recent transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding. However, reliable screens for mutants that improve protein stability do not yet exist, especially for proteins that are well folded and relatively stable.

Prediction of Residue-specific Contributions to Binding and Thermal Stability Using Yeast Surface Display.

Accurate prediction of residue burial as well as quantitative prediction of residue-specific contributions to protein stability and activity is challenging, especially in the absence of experimental structural information. This is important for prediction and understanding of disease causing mutations, and for protein stabilization and design. Using yeast surface display of a saturation mutagenesis library of the bacterial toxin CcdB, we probe the relationship between ligand binding and expression level of displayed protein, with solubility in and thermal stability.

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing.

Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single-residue resolution, using Cys labeling, viral neutralization assays, and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys-reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs.

COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a newly emerging, highly transmissible, and pathogenic coronavirus in humans that has caused global public health emergencies and economic crises. To date, millions of infections and thousands of deaths have been reported worldwide, and the numbers continue to rise. Currently, there is no specific drug or vaccine against this deadly virus; therefore, there is a pressing need to understand the mechanism(s) through which this virus enters the host cell.

Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling.

Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro.

Catalytic pathway, substrate binding and stability in SAICAR synthetase: A structure and molecular dynamics study.

The de novo purine biosynthesis is one of the highly conserved pathways among all organisms and is essential for the cell viability. A clear understanding of the enzymes in this pathway would pave way for the development of antimicrobial and anticancer drugs. Phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase is one of the enzymes in this pathway that catalyzes ATP dependent ligation of carboxyaminoimidazole ribotide (CAIR) with l-aspartate (ASP).

Molecular dynamics perspective on the protein thermal stability: a case study using SAICAR synthetase.

The enzyme SAICAR synthetase ligates aspartate with CAIR (5'-phosphoribosyl-4-carboxy-5-aminoimidazole) forming SAICAR (5-amino-4-imidazole-N-succinocarboxamide ribonucleotide) in the presence of ATP. In continuation with our previous study on the thermostability of this enzyme in hyper-/thermophiles based on the structural aspects, here, we present the dynamic aspects that differentiate the mesophilic (E. coli, E.

Structure of SAICAR synthetase from Pyrococcus horikoshii OT3: insights into thermal stability.

The first native crystal structure of Phosphoribosylaminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) from a hyperthermophilic organism Pyrococcus horikoshii OT3 was determined in two space groups H3 (Type-1: Resolution 2.35Å) and in C222(1) (Type-2: Resolution 1.9Å).

Differential activities of the two closely related withanolides, Withaferin A and Withanone: bioinformatics and experimental evidences.

Background And Purpose: Withanolides are naturally occurring chemical compounds. They are secondary metabolites produced via oxidation of steroids and structurally consist of a steroid-backbone bound to a lactone or its derivatives. They are known to protect plants against herbivores and have medicinal value including anti-inflammation, anti-cancer, adaptogenic and anti-oxidant effects.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3.

The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis.