title: A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins.
abstract: Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many processes in bacteria including biofilm formation, motility, and virulence (Hengge, 2009). Analysis of c-di-GMP binding properties of bacterial proteins is an important step to characterize c-di-GMP signaling pathways. C-di-GMP binds numerous proteins such as transcription factors, enzymes, and multimeric protein complexes (Hickman and Harwood, 2008, Ryjenkovet al., 2006, Weinhouseet al., 1997). The c-di-GMP binding assay described here is a relatively simple and cost effective method to characterize c-di-GMP binding to a protein using [32P]-labeled c-di-GMP. Radiolabeled c-di-GMP is readily synthesized with a purified GGDEF enzyme [such as WspR fromPseudomonas aeruginosa(P. aeruginosa)] and [a-32P]-GTP (Srivastavaet al.,2013). After incubation of the labeled c-di-GMP with the protein of interest in solution, the resulting mixture is filtered through a nitrocellulose protein binding membrane. The amount of labeled c-di-GMP that is retained on the membrane indicates the interaction between the signal and protein. The specificity of c-di-GMP binding can be tested by competing with unlabeled c-di-GMP or other nucleotides such as GTP in the reaction. By examining binding of a fixed protein concentration to increasing concentrations of c-di-GMP, this method is able to determine the dissociation constant of c-di-GMP-protein interaction.
authors: Department of Microbiology and Microbial Genetics, Michigan State University, East Lansing, USA.
authors: Disha
authors: D
authors: Srivastava
authors: Department of Microbiology and Microbial Genetics, Michigan State University, East Lansing, USA.
authors: Christopher M
authors: CM
authors: Waters