Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 88, Issue 2, Pages 229-236Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mimet.2011.11.015
Keywords
c-di-GMP-affinity pull down; Surface plasmon resonance; Chemical proteomics
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Funding
- Helmholtz-Gemeinschaft
- German Research Foundation (DFG) [SFB900]
- EU [222635]
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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. (C) 2011 Elsevier B.V. All rights reserved.
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