Analysis of c-di-GMP Levels Synthesized by a Photoreceptor Protein in Response to Different Light Qualities Using an In Vitro Enzymatic Assay

Methods Mol Biol. 2017;1657:187-204. doi: 10.1007/978-1-4939-7240-1_15.

Abstract

Diguanylate cyclases are enzymes that use two GTP molecules to produce one molecule cyclic dimeric guanosine monophosphate (c-di-GMP). This cyclic dinucleotide is an ubiquitous prokaryotic second messenger that controls a variety of cell functions. Several proteins have been described which contain a photoreceptor domain fused to a diguanylate cyclase. The cyanobacterial light sensor Cph2 is responsible for the blue-light induced synthesis of c-di-GMP in Synechocystis sp. PCC 6803. Here, we provide a detailed protocol for an in vitro enzymatic assay with a purified photoreceptor protein using light as the crucial reaction parameter for c-di-GMP synthesis. The assay is accomplished under continuous illumination with light of different quality with inactivation of the enzyme by heat denaturation. Analytics are performed using HPLC-UV.

Keywords: Cyclic diguanylate; Cyclic nucleotide; Enzyme-activity assay; In vitro enzymatic method; Light-dependent enzymatic activity; Photosensor; Photoswitch; Second messenger; c-di-GMP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism*
  • Chromatography, High Pressure Liquid
  • Cyanobacteria / physiology*
  • Cyanobacteria / radiation effects*
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / biosynthesis
  • Cyclic GMP / metabolism
  • Enzyme Activation
  • Enzyme Assays / methods
  • Enzyme Assays / standards
  • Light*
  • Reference Standards
  • Spectrophotometry, Ultraviolet

Substances

  • Bacterial Proteins
  • bis(3',5')-cyclic diguanylic acid
  • Cyclic GMP