Purification and assays of Rhodobacter capsulatus RegB-RegA two-component signal transduction system.
Two-component signal-transduction systems, composed of a histidine-sensor kinase and a DNA-binding response regulator, allow bacteria to detect environmental changes and adjust cellular physiology to live more efficiently in a broad distribution of niches. Although many two-component signal-transduction systems are known, a limited number of signals that stimulate these systems have been discovered. This chapter describes the purification and characterization of the predominant two-component signal-transduction system utilized by Rhodobacter capsulatus, a nonsulfur purple photosynthetic bacterium. Specifically, we explain the overexpression, detergent solubilization, and purification of the full-length membrane-spanning histidine-sensor kinase RegB. We also provide a method to measure autophosphorylation of RegB and discern the effect of its signal molecule, ubiquinone, on autophosphorylation levels. In addition we describe the overexpression and purification of the cognate response regulator RegA and a technique used to visualize the phosphotransfer reaction from RegB to RegA.
Swem
Lee R
LR
Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.
Swem
Danielle L
DL
Wu
Jiang
J
Bauer
Carl E
CE
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D017931:DNA Primers; D015964:Gene Expression Regulation, Bacterial; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*
D001426:Bacterial Proteins; D017931:DNA Primers; C091084:RegA protein, Rhodobacter; C091081:RegB protein, Rhodobacter; D015534:Trans-Activators; D011494:Protein Kinases