A simple and sensitive detection of the binding ligands by using the receptor aggregation and NMR spectroscopy: a test case of the maltose binding protein

Abstract

Protein-ligand interaction is one of the highlights of molecular recognition. The most popular application of this type of interaction is drug development which requires a high throughput screening of a ligand that binds to the target protein. Our goal was to find a binding ligand with a simple detection, and once this type of ligand was found, other methods could then be used to measure the detailed kinetic or thermodynamic parameters. We started with the idea that the ligand NMR signal would disappear if it was bound to the non-tumbling mass. In order to create the non-tumbling mass, we tried the aggregates of a target protein, which was fused to the elastin-like polypeptide. We chose the maltose binding proteinas a test case, and we tried it with several sugars, which included maltose, glucose, sucrose, lactose, galactose, maltotriose, and β-cyclodextrin. The maltose signal in the H-1 NMR spectrum disappeared completely as hoped around the protein to ligand ratio of 1:3 at 298 K where the proteins aggregated. The protein signals also disappeared upon aggregation except for the fast-moving part, which resulted in a cleaner background than the monomeric form. Since we only needed to look for a disappearing signal amongst those from the mixture, it should be useful in high throughput screening. Other types of sugars except for the maltotriose and β-cyclodextrin, which are siblings of the maltose, did not seem to bind at all. We believe that our system would be especially more effective when dealing with a smaller target protein, so both the protein and the bound ligand would lose their signals only when the aggregates formed. We hope that our proposed method would contribute to accelerating the development of the potent drug candidates by simultaneously identifying several binders directly from a mixture.

Identification of interaction partners using protein aggregation and NMR spectroscopy

“…1D 1 H NMR experiments will be employed [30,31]. The previous experience suggested that the 1D noesy (noesypr1d, Bruker pulseprogram) was quite suitable for the detection [22]. Fig 3 illustrates the process where the partner protein is released and the signal is restored (left), and the signal of the inhibitor disappears (right) as the inhibitor binds to the target protein.…”

“…We make the signals from the molecule of interest disappear if it binds to the target. In our previous report, we successfully materialized this concept in order to observe the disappearance of the signal of the binding ligand [22]. We wish to expand the application in order to probe the interaction between two proteins and its disruption by a ligand by observing disappearance and reappearance of the signals from the protein of interest.…”