Activity

Included are procedures for the production and purification of recombinant PHD2, followed by investigations into PHD2-catalyzed reactions using mass spectrometry, and studies on the stabilization of HIF in cells employing immunoblotting techniques.

Recently identified as a member of the mammalian globin family, androglobin (ADGB) is a chimeric protein. A circularly permuted, embedded globin domain within this protein exhibits a hexacoordinated heme-binding pattern. Despite initial findings of ADGB’s restricted expression to cells in the postmeiotic phases of spermatogenesis, RNA-Seq analysis indicates its presence in cells characterized by motile cilia or flagella. The rigorous control exerted over ADGB gene expression necessitates the introduction of alternative methods to investigate its inherent expression in typical mammalian cell lines which lack expression of ADGB. This report details the use of CRISPR activation (CRISPRa) for inducing endogenous ADGB gene expression in HEK293T, MCF-7, and HeLa cells, stemming from its promoter, and underscores its efficacy in confirming putative regulatory DNA elements of the ADGB gene located within both the promoter and enhancer regions.

Multicellular organisms exhibit evolved, complex strategies for the detection and accommodation of intracellular oxygen variations. The oxygen-sensing cellular pathway, comprised of the von Hippel-Lindau (pVHL) tumor suppressor protein, prolyl hydroxylases (PHD), and hypoxia-inducible factors (HIFs), regulates the expression of downstream genes vital for oxygen homeostasis. It has become increasingly apparent in recent years that the regulation of oxygen levels is deeply entwined with the cellular pathways that detect and respond to iron. The activities of prolyl-hydroxylases, E3 ubiquitin ligase adaptor protein FBXL5, iron regulatory proteins (IRPs), and Fe-S cluster proteins, integral components of these networks, are critically dependent on both iron and oxygen, or are tightly regulated by intracellular quantities of these essential elements. The remodeling of protein interactomes, contingent on intracellular oxygen and iron levels, provides insights into the intricacies and dynamics of these pathways. Recently, an oxygen-sensitive interaction between FBXL5 and the cytoplasmic iron-sulfur cluster targeting complex (CIA targeting complex) was documented, impacting the FBXL5-dependent regulation of IRPs. pi3k signals receptor From this research, we present a protocol to establish and sustain hypoxic conditions in cultured mammalian cells, along with a mass spectrometry-based proteomics strategy designed to scrutinize the interactome alterations of vital proteins, as influenced by intracellular oxygen and iron. Iron and oxygen signaling dynamics are readily understood using these broadly applicable methods.

To catalyze intricate biological reactions, nonheme diiron enzymes utilize the chemical potential of oxygen. The enzymes’ dormant state involves a diferrous cofactor, bound and coordinated by histidine and carboxylate ligands. The cofactor, subjected to oxygen, undergoes oxidation to its diferric state, forming a peroxo-intermediate. This intermediate then facilitates diverse oxidative reactions, including desaturation and heteroatom oxidation. In spite of their adaptability and effectiveness, a novel group of non-heme diiron enzymes displays inherent instability in their cofactors, thereby resisting structural elucidation. Among the members of the heme-oxygenase-like diiron oxidase/oxygenase (HDO) superfamily, this feature is found commonly. HDOs exhibit a flexible core structure, which dynamically remodels upon the addition of a metal Although a substantial number, approximately 9600, of HDOs have been found, their functional characterization remains limited to a few cases up to now. The chapter describes the techniques employed to characterize the HDO N-oxygenase, specifically SznF. The experimental procedure for overexpression and purification of apo-SznF is detailed, encompassing a specialized method to facilitate the attainment of an X-ray structure of the holo-SznF protein. Employing stopped-flow absorption and Mossbauer spectroscopies, we further detail the characterization of the transient SznF-peroxo-Fe(III)2 complex. By utilizing these studies, a framework for characterizing novel members of the HDO superfamily is created.

Protein dynamics and interactions are studied via the established hydrogen/deuterium exchange (HDX) approach, an analytical technique that examines the isotope exchange of backbone amides. With virtually no constraints on protein dimensions, adaptability, or experimental parameters, this process can be undertaken in solution under varied pH, temperature, and controlled redox settings. The method’s combination with mass spectrometry (MS) results in straightforward execution and high throughput, making it an excellent complement to high-resolution structural biology techniques. Given the recent advancements in artificial intelligence-based protein structure modeling, there is a considerable need for techniques that allow for swift and unambiguous validation of predictions generated in silico; hydrogen/deuterium exchange mass spectrometry (HDX-MS) is particularly adept at addressing this requirement. Employing HDX-MS, we delineate a protocol and demonstrate its effectiveness in examining the dynamic adjustments and structural modifications of a dimeric heme-containing oxygen-sensing protein experiencing changes in its coordination and oxidation states. Consequently, a mechanism for transducing the signal (oxygen binding to the heme iron in the sensing domain) to the functional domain of the protein was proposed.

A spectrophotometric approach for assessing the breakdown of the bacterial second messenger cyclic dimeric guanosine monophosphate is presented, enabling the characterization of enzymes under conditions ranging from aerobic to anaerobic. This method extracts all required data to calculate KM and kcat from reactions, using a single 96-well plate measured by a standard plate reader. Employing a spectrophotometric assay, the rates and Michaelis-Menten parameters of the c-di-GMP phosphodiesterase DcpG were determined, considering its sensor domain in various ligation states.

Gaseous ligand variations in the bacteria’s environment trigger a spectrum of behaviors, including the synthesis of the secondary messenger cyclic di-GMP. Measuring gas sensing can be challenging due to the abundance of oxygen in the atmosphere, especially within systems sensitive to oxidation-reduction reactions. We present an anaerobic technique for evaluating cyclic di-GMP synthesis, examining the impact of oxygen, nitric oxide, and carbon monoxide on a diguanylate cyclase’s activity and potential experimental shortcomings.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) incorporating Phos-tag (Phos-tag electrophoresis), a non-radioactive technique, is used to assess the autophosphorylation of a kinase and/or its transfer of phosphate to a substrate protein from the kinase/ATP complex. Compared to radioisotope methods utilizing [32P]ATP, this technique achieves a higher degree of sensitivity in the detection of minute quantities of phosphorylated protein in fresh protein samples. In-depth investigations into the kinase activity of a heme-based oxygen sensor—specifically, a globin-coupled histidine kinase found in the soil bacterium Anaeromyxobacter sp.—relied on phos-tag electrophoresis. Fw109-5 (AfGcHK), a specific model, is specified.

FeFe hydrogenases, a vital class of hydrogen-evolving enzymes, often demonstrate oxygen sensitivity and consequently require anaerobic conditions for their scientific analysis and characterization. For elucidating the reaction mechanisms of the complex six-iron active center of [FeFe] hydrogenases, specifically the H-cluster, understanding the electrochemical interactions between different active and inactive states of these enzymes is paramount. The distinctive spectral fingerprints of H-cluster states in the mid-IR region enable IR spectroelectrochemical experiments to provide a powerful methodological framework for this purpose. Spectroelectrochemical experiments employing Fourier-transform infrared (FTIR) spectroscopy are outlined in this chapter for investigating [FeFe] hydrogenases under anaerobic conditions. A study of experimental design, data acquisition, and data analysis formed a significant part of the curriculum.

A substantial collection of vital in vivo mechanisms and pathways are interwoven with the diverse roles performed by a multitude of oxygen-binding heme proteins. To gain a structural understanding of these proteins, Resonance Raman (rR) spectroscopy is an excellent technique, revealing information on the geometry of the Fe-O-O fragment and its electrostatic interactions within the distal active site. The task of characterizing these oxy adducts is significantly hindered by their instability, notably in heme protein solutions, an obstacle surmountable through employing the rR technique on cryogenically frozen specimens. This report provides a comprehensive explanation of the technique for determining the rR spectra of heme proteins with stable oxygenated states, and the necessary technical alterations for measuring unstable samples held at 77 Kelvin.

The proliferation of proteins that function in both gaseous ligand sensing and detoxification has spurred renewed appreciation for hemeproteins. The importance of measuring the binding affinities of these proteins for ligands like oxygen, carbon monoxide, and nitric oxide is undeniable. Precisely defining full ligand saturation and estimating competitive ligand displacement are also critical aspects. The acquisition protocol for an intact O2-binding hemeprotein with a complete complement of heme is provided, including analysis of factors that influence its oxygen binding affinity, and precise determination of equilibrium and kinetic parameters, including Kd, kon, and koff for oxygen binding.

In protein-based oxygen sensors, affinity values exhibit a considerable spectrum, spanning the low nanomolar to the high micromolar range. The intricate interplay of metals, cofactors, and macromolecular structures in proteins, governing their oxygen affinity (Kd), is crucial for their biological function, a significant area of inquiry in biochemistry and microbiology.