Discovery of the Xenon–Protein Interactome Using Large-Scale Measurements of Protein Folding and Stability

Abstract

The intermolecular interactions of noble gases in biological systems are associated with numerous biochemical responses, including apoptosis, inflammation, anesthesia, analgesia, and neuroprotection. The molecular modes of action underlying these responses are largely unknown. This is in large part due to the limited experimental techniques to study protein–gas interactions. The few techniques that are amenable to such studies are relatively low-throughput and require large amounts of purified proteins. Thus, they do not enable the large-scale analyses that are useful for protein target discovery. Here, we report the application of stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) methodologies to detect protein–xenon interactions on the proteomic scale using protein folding stability measurements. Over 5000 methionine-containing peptides and over 5000 semi-tryptic peptides, mapping to ∼1500 and ∼950 proteins, respectively, in the yeast proteome, were assayed for Xe-interacting activity using the SPROX and LiP techniques. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known to bind Xe. A bioinformatics analysis of the proteomic results revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes. A fraction of the protein targets that were identified are tied to previously established modes of action related to xenon’s anesthetic and organoprotective properties. These results enrich our knowledge and understanding of biologically relevant xenon interactions. The sample preparation protocols and analytical methodologies developed here for xenon are also generally applicable to the discovery of a wide range of other protein–gas interactions in complex biological mixtures, such as cell lysates.

Radical-Mediated Covalent Azidylation of Hydrophobic Microdomains in Water-Soluble Proteins

“…H 2 O 2 was selected as the oxidant due to its relative ease of use, availability, and reported oxidizing capability in a protein context. 29,30 A publication that reports the addition of azido radicals to free tryptophan suggests that the adduct reaction is inefficient and produces a product with relatively low abundance. 17 Thus, we developed an assay to detect and enrich the modified compound via click chemistry (CuAAC).…”

“…Classically, azido radicals that interact with free amino acids are generated by linear particle accelerators irradiating water to create hydroxyl radicals, which in turn react with azide to produce azido radicals. , Given this, we asked whether more accessible methods of oxidation could facilitate azido radical generation in a physiological buffer system. H 2 O 2 was selected as the oxidant due to its relative ease of use, availability, and reported oxidizing capability in a protein context. , A publication that reports the addition of azido radicals to free tryptophan suggests that the adduct reaction is inefficient and produces a product with relatively low abundance . Thus, we developed an assay to detect and enrich the modified compound via click chemistry (CuAAC) .…”

“…Recent advances have been made that greatly expand the utility of covalent labeling reagents for mapping interacting surfaces and for reporting on conformational changes in soluble proteins. ,− For example, new reagents have been developed that can react with aromatic sidechains within membrane proteins and on hydrophobic patches, − and a handful of recently developed methods are aimed at measuring proteome-wide conformational changes. − In this report, while we set out to exploit the azido radical as a simpler means of general covalent protein labeling and subsequent derivatization, the product of these experiments is also a method for covalently modifying protein with the azido radical that captures and identifies locations of noncovalent azide binding. With our method, azide may present a unique, enrichable method to add to the existing toolbox.…”

Profiling protein targets of cellular toxicant exposure

“…SPROX is the most mature of the technologies used for characterizing protein structure proteome-wide, having been applied to numerous systems over the course of many years, and benchmarked against alternative technologies. [126][127][128][129][130] SPROX is well-validated for providing reliable unfolding free energies for at least proteins that follow twostate unfolding models. Even for proteins that defy two-state transitions, oxidation kinetics are a valid proxy for destabilization.…”

Stability-Based Proteomics for Investigation of Structured RNA–Protein Interactions

“…The peptide-based readouts in SPROX, LiP, PP, and DARTS provide structural information about the sites of ligand-induced conformational changes in proteins, whereas the protein-based readouts in TPP and CPP preclude the acquisition of such information. All of the above techniques have been widely used to identify the protein targets of drugs ,− and other biologically relevant molecules. ,,,− It has been found that using more than one of the above proteomics methods for protein target discovery can reduce the false positive rate as overlapping hits are more likely to be true positives . Although these methods have been widely used in protein target elucidation studies, they have not been used to study structured RNA–protein interactions.…”