The Differential Radial Capillary Action of Ligand Assay Enables Systematic Identification and Characterization of Ligand Binding Interactions with Proteins and Nucleic Acids Abstract

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Cyclic di-GMP (cdiGMP) is a ubiquitous prokaryotic nucleotide signaling molecule that regulates important bacterial processes, including biofilm formation, motility, and virulence. The differential radial capillary action of ligand assay (DRaCALA) was developed to determine protein-ligand interactions and provide insight into the mechanism of cdiGMP signal transduction. DRaCALA is based on the ability of nitrocellulose membranes to separate free ligand from bound protein-ligand complexes resulting in precise measurements of the fraction of bound ligand. The principle of DRaCALA was demonstrated by detection of 3 radiolabeled nucleotides binding to their cognate receptors. DRaCALA also enabled the determination of affinity, specificity, and kinetics of cdiGMP-binding to Alg44. A unique feature of DRaCALA is the ability to determine specific binding interactions to heterologously expressed proteins in whole cell lysates, suggesting that individual open reading frames could be screened for the expression of a cdiGMP-binding protein. DRaCALA was applied on a genome-wide scale to systematically screen protein products of over 98% of Vibrio cholerae open reading frames for cdiGMP-binding activity. The DRaCALA ORFeome screen identified 5 of 10 previously described cdiGMP-binding proteins, 19 proteins with predicted cdiGMP-binding domains, and 6 novel putative cdiGMP-binding proteins lacking a defined cdiGMP-binding site. Direct cdiGMP-binding was demonstrated for the T2SE ATPase MshE, and cdiGMP-binding activity was observed for a Pseudomonas aeruginosa homolog PA14_29490. These results suggest that cdiGMP-binding may be a conserved feature of a subset of T2SE ATPases that regulate type IV pili and type II secretion. Finally, the applications of DRaCALA were extended for the determination of protein binding to nucleotide polymers of DNA and RNA and cdiGMP-binding to a RNA aptamer. In total these studies report the development of DRaCALA as a novel biochemical assay and its use in the systematic identification and characterization of protein-ligand interactions.