title: A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa.
abstract: In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2'-aminohexylcarbamoyl-c-di-GMP (2'-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2'-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.
authors.affiliation: Department of Cell Biology, Helmholtz Center for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Düvel
authors.forename: Daniela
authors.initials: D
authors.lastname: Bertinetti
authors.forename: Stefan
authors.initials: S
authors.lastname: Möller
authors.forename: Frank
authors.initials: F
authors.lastname: Schwede
authors.forename: Michael
authors.initials: M
authors.lastname: Morr
authors.forename: Josef
authors.initials: J
authors.lastname: Wissing
authors.forename: Lena
authors.initials: L
authors.lastname: Radamm
authors.forename: Bastian
authors.initials: B
authors.lastname: Zimmermann
authors.forename: Hans-Gottfried
authors.initials: HG
authors.lastname: Genieser
authors.forename: Lothar
authors.initials: L
authors.lastname: Jänsch
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
authors.forename: Susanne
authors.initials: S
authors.lastname: Häussler
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP