title: Detection of cyclic diguanylate G-octaplex assembly and interaction with proteins.
abstract: Bacterial signaling networks control a wide variety of cellular processes including growth, metabolism, and pathogenesis. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (cdiGMP) is a secondary signaling nucleotide that controls cellulose synthesis, biofilm formation, motility and virulence in a wide range of gram-negative bacterial species. CdiGMP is a dynamic molecule that forms different tertiary structures in vitro, including a trans-monomer, cis-monomer, cis-dimer and G-octaplex (G8). Although the monomer and dimer have been shown to be physiologically relevant in modulating protein activity and transcription, the biological effects of the cdiGMP G8 has not yet been described. Here, we have developed a TLC-based assay to detect radiolabeled cdiGMP G8 formation. Utilizing the radiolabeled cdiGMP G8, we have also shown a novel inhibitory interaction between the cdiGMP G8 and HIV-1 reverse transcriptase and that the cdiGMP G8 does not interact with proteins from Pseudomonas aeruginosa known to bind monomeric and dimeric cdiGMP. These results suggest that the radiolabeled cdiGMP G8 can be used to measure interactions between the cdiGMP G8 and cellular proteins, providing an avenue through which the biological significance of this molecule could be investigated.
authors: Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, College Park, Maryland, United States of America.
authors: Ori J
authors: OJ
authors: Lieberman
authors: Jeffrey J
authors: JJ
authors: DeStefano
authors: Vincent T
authors: VT
authors: Lee
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002855:Chromatography, Thin Layer; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D058105:Polymerization; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D015398:Signal Transduction; D012996:Solutions; D013194:Staining and Labeling; D013237:Stereoisomerism
chemical_list: D001426:Bacterial Proteins; D010761:Phosphorus Radioisotopes; D012996:Solutions; C062025:bis(3',5')-cyclic diguanylic acid; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D006152:Cyclic GMP