Monitoring the Kinase Activity of Heme-Based Oxygen Sensors and Its Dependence on O2 and Other Ligands Using Phos-Tag Electrophoresis.

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).

Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins.

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.

Characterization of the interaction between the tumour suppressor p53 and heme and its role in the protein conformational dynamics studied by various spectroscopic techniques and hydrogen/deuterium exchange coupled with mass spectrometry.

The tumour suppressor p53 regulates the expression of a myriad of proteins that are important for numerous cellular processes, including apoptosis, cell cycle arrest, DNA repair, metabolism, and even autophagy and ferroptosis. Aside from DNA, p53 can interact with many types of partners including proteins and small organic molecules. The ability of p53 to interact with heme has been reported so far. In this study, we used various spectroscopic studies to conduct a thorough biophysical characterization of the interaction between p53 and heme concerning the oxidation, spin, coordination, and ligand state of heme iron. We found that the p53 oligomeric state and zinc biding ability are preserved upon the interaction with heme. Moreover, we described the effect of heme binding on the conformational dynamics of p53 by hydrogen/deuterium exchange coupled with mass spectrometry. Specifically, the conformational flexibility of p53 is significantly increased upon interaction with heme, while its affinity to a specific DNA sequence is reduced by heme. The inhibitory effect of DNA binding by heme is partially reversible. We discuss the potential heme binding sites in p53 with respect to the observed conformational dynamics changes and perturbed DNA-binding ability of p53 upon interaction with heme.

Signal transduction mechanisms in heme-based globin-coupled oxygen sensors with a focus on a histidine kinase (AfGcHK) and a diguanylate cyclase (YddV or EcDosC).

Heme is a vital cofactor of proteins with roles in oxygen transport (e.g. hemoglobin), storage (e.g. myoglobin), and activation (e.g. P450) as well as electron transfer (e.g. cytochromes) and many other functions. However, its structural and functional role in oxygen sensing proteins differs markedly from that in most other enzymes, where it serves as a catalytic or functional center. This minireview discusses the mechanism of signal transduction in two heme-based oxygen sensors: the histidine kinase AfGcHK and the diguanylate cyclase YddV (EcDosC), both of which feature a heme-binding domain containing a globin fold resembling that of hemoglobin and myoglobin.