Abstract
Small molecules not only represent cellular building blocks and metabolic intermediates, but also regulatory ligands and signaling molecules that interact with proteins. Although these interactions affect cellular metabolism, growth, and development, they have been largely understudied. Herein, we describe a method, which we named PROtein–Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein–small molecule and of protein–protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Proteins and small molecules showing similar elution behavior, across protein-containing fractions, constituted putative interactors. Applying PROMIS to an A. thaliana extract, we ascertained known protein–protein (PPIs) and protein–metabolite (PMIs) interactions and reproduced binding between small-molecule protease inhibitors and their respective proteases. More importantly, we present examples of two experimental strategies that exploit the PROMIS dataset to identify novel PMIs. By looking for similar elution behavior of metabolites and enzymes belonging to the same biochemical pathways, we identified putative feedback and feed-forward regulations in pantothenate biosynthesis and the methionine salvage cycle, respectively. By combining PROMIS with an orthogonal affinity purification approach, we identified an interaction between the dipeptide Tyr–Asp and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. In summary, we present proof of concept for a powerful experimental tool that enables system-wide analysis of PMIs and PPIs across all biological systems. The dataset obtained here comprises nearly 140 metabolites and 5000 proteins, which can be mined for putative interactors.
Highlights
Small molecules represent cellular building blocks and metabolic intermediates, and regulatory ligands and signaling molecules that interact with proteins
We have shown that size-exclusion chromatography (SEC) can be used to separate small-molecule–protein complexes based on their molecular weight
Protein and smallmolecule complexes were separated by size-exclusion chromatography (SEC)
Summary
Small molecules represent cellular building blocks and metabolic intermediates, and regulatory ligands and signaling molecules that interact with proteins. We describe a method, which we named PROtein–Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein–small molecule and of protein–protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Small molecules represent cellular building blocks and metabolic intermediates and regulatory ligands and signaling molecules, exerting their functions via interaction with macromolecules, most commonly proteins. The most promising approaches include the following: 1) purification and characterization of protein–metabolite complexes using affinity-tagged protein baits [4, 5]; 2) identification of protein partners using small molecules as affinity baits [6]; 3) drugaffinity–responsive target stability assay (DARTS) [7] and thermal proteome profiling [8, 9], both exploiting differences in the stability between unbound and small-molecule– bound proteins to find protein targets of drug compounds; and 4) chemoproteomic methods [10], taking advantage of chemically modified small molecules, which upon binding covalently label their protein partners
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.