Oxygen-Sensing Histidine-Protein Kinases: Assays of Ligand Binding and Turnover of Response-Regulator Substrates

Marie Alda Gilles-Gonzalez, Gonzalo Gonzalez, Eduardo Henrique Silva Sousa, Jason Tuckerman

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Heme-based sensors are a recently discovered functional class of heme proteins that serve to detect physiological fluctuations in oxygen (O2), carbon monoxide (CO), or nitric oxide (NO). Many of these modular sensors detect heme ligands by coupling a histidine-protein kinase to a heme-binding domain. They typically bind O2, CO, and NO but respond only to one of these ligands. Usually, they are active in the ferrous unliganded state but are switched off by saturation with O2. The heme-binding domains of these kinases are quite varied. They may feature a PAS fold, as in the Bradyrhizobium japonicum and Sinorhizobium melitoti FixL proteins, or a GAF fold, as in the Mycobacterium tuberculosis DevS and DosT proteins. Alternative folds, such as HNOB (also H-NOX), have also been noted for such signal-transducing kinases, although these classes are less well studied. Histidine-protein kinases function in partnership with cognate response-regulator substrate(s): usually transcription factors that they activate by phosphorylation. For example, FixL proteins specifically phosphorylate their FixJ partners, and DevS and DosT proteins phosphorylate DevR in response to hypoxia. We present methods for purifying these sensors and their protein substrates, verifying the quality of the preparations, determining the Kd values for binding of ligand and preparing sensors of known saturation, and measuring the rates of turnover (kcat) of the protein substrate by sensors of known heme status.

Original languageEnglish (US)
Pages (from-to)173-189
Number of pages17
JournalMethods in Enzymology
Volume437
DOIs
StatePublished - 2008

Fingerprint

Heme
Histidine
Protein Kinases
Assays
Oxygen
Ligands
Sensors
Substrates
Carbon Monoxide
Proteins
Nitric Oxide
Sinorhizobium
Phosphotransferases
Bradyrhizobium
Hemeproteins
Phosphorylation
Transcription Factors
Histidine Kinase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Oxygen-Sensing Histidine-Protein Kinases : Assays of Ligand Binding and Turnover of Response-Regulator Substrates. / Gilles-Gonzalez, Marie Alda; Gonzalez, Gonzalo; Sousa, Eduardo Henrique Silva; Tuckerman, Jason.

In: Methods in Enzymology, Vol. 437, 2008, p. 173-189.

Research output: Contribution to journalArticle

Gilles-Gonzalez, Marie Alda ; Gonzalez, Gonzalo ; Sousa, Eduardo Henrique Silva ; Tuckerman, Jason. / Oxygen-Sensing Histidine-Protein Kinases : Assays of Ligand Binding and Turnover of Response-Regulator Substrates. In: Methods in Enzymology. 2008 ; Vol. 437. pp. 173-189.
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