A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa

• Published in: Journal of microbiological methods 2012-02, Vol.88 (2), p.229-236
• Main Authors: Düvel, Juliane, Bertinetti, Daniela, Möller, Stefan, Schwede, Frank, Morr, Michael, Wissing, Josef, Radamm, Lena, Zimmermann, Bastian, Genieser, Hans-Gottfried, Jänsch, Lothar, Herberg, Friedrich W., Häussler, Susanne
• Format: Article
• Language: English
• Subjects: bacteria; Bacterial Proteins - analysis; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacteriological methods and techniques used in bacteriology; Bacteriology; binding proteins; biofilm; Biological and medical sciences; c-di-GMP-affinity pull down; Carrier Proteins - analysis; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Chemical proteomics; Cyclic GMP - analogs & derivatives; Cyclic GMP - chemistry; Cyclic GMP - metabolism; Fundamental and applied biological sciences. Psychology; mass spectrometry; Microbiology; pathogens; proteomics; Proteomics - methods; Pseudomonas aeruginosa; Pseudomonas aeruginosa - chemistry; Pseudomonas aeruginosa - metabolism; Signal Transduction; Surface Plasmon Resonance
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2011.11.015

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. ► We immobilize functionalized c-di-GMP on a solid support. ► This affinity matrix serves the isolation of c-di-GMP binding proteins. ► MS-identified proteins harbor known but also new c-di-GMP binding motifs. ► We establish surface plasmon resonance assays to validate c-di-GMP binding. ► The biochemical tools will help to understand the complex c-di-GMP signaling system.