Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.

• Keywords
• Globin-coupled histidine kinase, Heme-containing oxygen sensors, Hydrogen/deuterium exchange, Ligand binding, Mass spectrometry, Protein conformational dynamics, Signal transduction,
• MeSH
• Deuterium MeSH
• Heme chemistry   MeSH
• Hemeproteins * MeSH
• Mass Spectrometry methods   MeSH
• Oxygen metabolism   MeSH
• Artificial Intelligence MeSH
• Deuterium Exchange Measurement methods   MeSH
• Hydrogen chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Heme MeSH
• Hemeproteins * MeSH
• Oxygen MeSH
• Hydrogen MeSH

The nonradioactive method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Phos-tag (Phos-tag electrophoresis), is used to evaluate a kinase autophosphorylation and/or phosphotransfer reaction from a kinase/ATP to its protein substrate. This method outperforms radioisotope methods using [32P]ATP for detecting trace amounts of phosphorylated protein in fresh protein preparations. Phos-tag electrophoresis has been used to perform detailed analyses of the kinase activity of a heme-based oxygen sensor-specifically, a globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 (AfGcHK).

• Keywords
• Autophosphorylation reaction, Heme-based oxygen sensors, Kinase activity, Ligand binding, Ligand-dependent kinase activity, Phos-tag electrophoresis, Phosphotransfer reaction,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Bacteria metabolism   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Heme * metabolism   MeSH
• Oxygen metabolism   MeSH
• Ligands MeSH
• Proteins * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol MeSH Browser
• Adenosine Triphosphate MeSH
• Heme * MeSH
• Oxygen MeSH
• Ligands MeSH
• Proteins * MeSH

The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.

• Keywords
• bacterial protein kinase, cell signaling, crystal structure, dimerization interface, globin, heme, heme-based oxygen sensor, histidine kinase, signal transduction, two component system,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Globins chemistry   metabolism   MeSH
• Histidine Kinase chemistry   metabolism   MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Myxococcales chemistry   metabolism   MeSH
• Protein Domains MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Globins MeSH
• Histidine Kinase MeSH