A binding agreement: Folding changes reveal protein-drug interactions

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  • Published: Oct 5, 2012
  • Author: Steve Down
  • Channels: Proteomics & Genomics / Proteomics
thumbnail image: A binding agreement: Folding changes reveal protein-drug interactions

Protein-drug binding

Proteins targeted by particular drugs and metabolites can be identified by a simple process in which the effects on protein unfolding are measured by SDS-PAGE following proteolytic digestion.

Finding a drug that works is only part of the battle these days. Knowing the molecular target of the drug within the body is essential in order to reveal how it works and help to develop compounds with improved pharmacological properties. With so many potential drugs under development in libraries, discovering the drug-target relationship has the potential to become a bottleneck in the development process.

Recently, a new approach for identifying proteins that bind with a particular drug candidate was reported. Proteins will generally unfold at different rates in the bound form compared with the free form, so observing a change in protein conformation is a firm indication of binding to another compound. This “energetics-based” approach does not require the use of labelled compounds and is carried out in solution, so does not require immobilisation as in affinity binding methods.

In 2011, a team of scientists in the USA published details of a new procedure for monitoring the stability changes of proteins upon binding. Chiwook Park and colleagues from Purdue University, West Lafayette, used pulse proteolysis which is intended to digest only the unfolded proteins in an equilibrium mixture of folded and unfolded proteins. The mixtures were pre-fractionated by 2D gel electrophoresis and each fraction was subjected to pulse proteolysis.

In their improved method, the researchers replaced 2D gel electrophoresis by anion exchange chromatography and 1D SDS-PAGE, which together provide comparable resolution to 2D gel electrophoresis but result in a simpler quantitative method with better reproducibility. Writing in Protein Science, Park illustrated the method by searching for proteins in Escherichia coli that bind to ATP.

Pulse proteolysis

In the first stage, a soluble lysate of E. coli was fractionated on an anion exchange column to give 15 separate fractions which were each subjected to pulse proteolysis. They were mixed with dithiothreitol and varying concentrations of urea to induce unfolding of the proteins in the presence and absence of ATP. In practice, adenosine 5′-[γ-thio]triphosphate was used in place of ATP to give better resistance to hydrolysis.

After standing overnight to allow binding and unfolding to occur, the mixtures were incubated with thermolysin. The resulting digest was quenched to stop the reaction before the mixture was subjected to 1D SDS-PAGE. The intensities of each protein band in the absence and presence of ATP were measured from the electropherograms and used to calculate the binary logarithm of the intensity ratios. Proteins with values greater than 50% were said to undergo significant binding with ATP and were identified by in-gel digestion and MALDI mass spectrometry.

Novel ATP-binding proteins

The combination of anion exchange chromatography and 1D SDS-PAGE led to an even distribution of proteins throughout the 15 fractions with about 30-50 proteins in each. Pulse proteolysis then identified proteins that were unfolded in the absence of ATP and folded in its presence. Folding and unfolding occur at different urea concentrations for particular proteins, so a range of concentrations were applied to each fraction.

A total of 30 ATP-binding proteins were identified, including 9 that were previously not known to interact with ATP. The biochemical functions of the new ones are all well-known and none require ATP, so its role remains unclear at present. It may be that ATP has a regulatory function or that some of the proteins are multi-functional, requiring ATP for an extra function. Six of the nine proteins are known to bind to uridine, GTP, FAD or NAD+, which are similar ligands to ATP and it is possible that ATP inhabits their binding sites.

One of the novel ATP binders is phosphoglyceromutase and it was selected for further studies to confirm that the binding experiments are producing true results. The protein was overexpressed and purified from E. coli and subjected to pulse proteolysis in the absence and presence of ATP and varying concentrations of urea. The results clearly showed that ATP increased the stability of the protein by binding to it.

The new method is better than conventional approaches to protein-ligand binding because the compounds need no modification or isotope labelling and no immobilisation is required. It can also easily be extended to study multiple ligands. The replacement of 2D gel electrophoresis by LC and 1D SDS-PAGE reduces experimental complexity and improves reproducibility.

Related Links

Protein Science 2012, 21, 1280-1287: "Simplified proteomics approach to discover protein-ligand interactions"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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