Protein interactions with low molecular weight ligand have great implication in biology for both allosteric regulation and enzymatic activity. Another important aspect of protein-ligand interaction is for the development of pharmaceuticals and their intended and non-specific effects on biological systems. However, current technology has been limited to specific assays that are difficult to adapt to high-throughput screening to allow identification of novel protein receptors and small molecule inhibitors. To bypass these limitations, we have developed a novel assay based on differential radial capillary action. Initial work has demonstrated the differential radial capillary assay can detect the interaction between a bacterial secondary signaling molecule, cyclic-di-GMP (cdiGMP) and the Alg44 receptor protein. The assay allows detection of specificity based on competition experiments with unlabeled ligands. In addition, the differential radial capillary assay allows for quantitative measurements of dissociation constant and dissociation rate of cdiGMP with Alg44 which matches previously published reports. Furthermore, the assay can detect the interaction of cdiGMP with whole cell lysates from cells expressing the Alg44 receptor. The goals of this proposal are to explore the limitations of the differential radial capillary assay and the applicability of the assay for high-throughput screening for protein-ligand interactions.
In Aim 1, we will determine the specificity and accuracy of the differential radial capillary assay for other protein-ligand pairs, the range of compatible ligands, the ability to detect biochemical reactions and the solubility requirement for heterologously expressed proteins.
In Aim 2, we will investigate the limit of detection of the assay in whole cell system and determine if other model protein expressing systems, such as Saccharomyces cereviciae, insect cells and mammalian cells, are compatible with the assay. Furthermore, we will apply the assay to identify novel cdiGMP binding proteins by systematically screening the Pseudomonas aeruginosa and Vibrio cholerae ORFeomes. The results from these studies will have broad implication for the understanding of cdiGMP regulation and developing the field of functional metabolomics.
The ability to identify protein-ligand interactions is central to understanding regulation of biological systems as well as pharmaceutical treatment. This proposal seeks to develop a high-throughput differential radial capillary assay that can be used to analyze these interactions. Results from this work will provide a basis for determining the functional interactions between metabolites and drugs with their protein receptors.
|Roelofs, Kevin G; Jones, Christopher J; Helman, Sarah R et al. (2015) Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems. PLoS Pathog 11:e1005232|
|Orr, Mona W; Donaldson, Gregory P; Severin, Geoffrey B et al. (2015) Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover. Proc Natl Acad Sci U S A 112:E5048-57|
|Lieberman, Ori J; Orr, Mona W; Wang, Yan et al. (2014) High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases. ACS Chem Biol 9:183-92|
|Lieberman, Ori J; DeStefano, Jeffrey J; Lee, Vincent T (2013) Detection of cyclic diguanylate G-octaplex assembly and interaction with proteins. PLoS One 8:e53689|
|Donaldson, Gregory P; Roelofs, Kevin G; Luo, Yiling et al. (2012) A rapid assay for affinity and kinetics of molecular interactions with nucleic acids. Nucleic Acids Res 40:e48|
|Roelofs, Kevin G; Wang, Jingxin; Sintim, Herman O et al. (2011) Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions. Proc Natl Acad Sci U S A 108:15528-33|