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Many biological functions are facilitated by the interaction between two or more proteins. Studying and understanding protein-protein interactions is thus essential for understanding physiological processes in health and disease. Accordingly, protein science and interaction methods can be considered one of the most widespread applications in biologic and medicinal research and form one of the foundations of pharmacological theory. Modern microplate readers make it possible to record the entire extent of protein binding events.

Finding the best method to study protein-protein interactions

In recent years, the use of microplate readers has revolutionized the field of protein-protein interaction studies, not least due to the large number of possible measurement methods. Finding the right method for protein-protein interaction measurements is essential to assure high data quality and precise results. While some absorbance-based applications to study protein-protein interactions on microplate readers such as the sandwich ELISA are still in use, fluorescence- or luminescence-based measurements are more common nowadays. This is due to several advantages such as higher sensitivity as well as high-throughput compatibility. Here, detection methods based on Förster´s Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) are commonly employed. Both BRET and FRET are based on the energy transfer between two signal molecules. By attaching the two signal molecules to two proteins of interest, this signal transfer is used to detect their interaction. The binding interaction between the two proteins puts the attached signal molecules in close spatial proximity to each other, enabling the energy transfer (Figure 1).

BMG Labtech March 2024 Sponsored Content Figure 1
Figure 1. Example of a FRET assay with the pair CFP and YFP. A) Optical spectra of CFP and YFP: the CFP (donor) emission spectrum overlaps with the YFP (acceptor) excitation spectrum (spectral overlap). B) Schematic representation of FRET: in case of no interaction, CFP is excited and emits light at a specific wavelength. C) In case of interaction and FRET taking place, YFP additionally emits light at a different wavelength, and the emission intensity of CFP is quenched.

Advanced applications based on the principles of BRET and FRET have been developed to improve sensitivity and readout quality of protein-protein interaction measurements even further. NanoBRET uses NanoLuc® luciferase derived from deep sea shrimp as a signal molecule with highly increased signal output in comparison to conventional luciferases.

The following example based on a NanoBRET approach enabled the detection of the ubiquitination process of a protein. Ubiquitination labels proteins for degradation by cellular enzymes. This process is facilitated by proteolysis targeting chimeras (PROTACs). This property of PROTACs results in a special therapeutic potential for the targeted degradation of proteins implicated in disease. Here, the ubiquitination of the protein BRD4 was investigated in a kinetic assay. For this purpose, BRD4 was labelled with NanoLuc luciferase—using CRISPR/Cas-9 genome editing. In addition, single ubiquitin molecules were labelled with HaloTag fusion constructs coupled to a NanoBRET acceptor fluorophore. Ubiquitination of BRD4, arranged by the PROTAC ARV-771, put NanoLuc luciferase in close proximity to the labelled ubiquitin molecules and thereby enabled an increase of the BRET signal with ongoing ubiquitination in a concentration-dependent manner (Figure 2).

BMG Labtech March 2024 Sponsored Content Figure 2
Figure 2. NanoBRET assay monitoring the ubiquitination of the protein BRD4. BRET-based energy transfer between NanoLuc luciferase coupled to BRD4 and a fluorescent Halotag fusion construct linked to ubiquitin molecules increased during the ubiquitination process as ubiquitin gets into close spatial proximity to BRD4.

This application example again confirms the high sensitivity in detecting events at very low concentrations of PROTAC when using a NanoBRET-based assay.

BMG Labtech March 2024 Sponsored Content Figure 3
Figure 3. TR-FRET competition assay between CELT-335 (TR-FRET acceptor) and Δ9-THC,
Δ9-THCA and Δ9-THCV.

Alternatively to BRET, TR-FRET combines time-resolved measurements with the principle of FRET by using signal molecules with high signal stability over time. This allows the use of time windows to measure protein-protein interactions at which the interaction signal is still detectable while signals derived from assay background have mostly subsided, thereby improving sensitivity significantly. TR-FRET based assays are often applied to study the interaction of receptors with their ligands. In recent years, cannabinoid receptors, a subgroup of G protein-coupled receptors, have gained popularity. Next to phytocannabinoids with partly psychoactive effects, there are also endocannabinoids, which are formed in the human body and control important functions such as learning, pain and eating. Their role in these functions makes cannabinoids a potential tool to combat a variety of diseases, including neurological conditions, and drives efforts to understand the pharmacology of cannabinoids at the receptor level. The following application example deals with differential binding of ∆9-tetrahydrocannabinol derivatives to the cannabinoid receptor 1 (CB1). For the interaction study, CB1 was labelled with the TR-FRET donor terbium while the known CB1 ligand CELT-335 is also fluorescently labelled. Upon the binding of the labelled ligand to CB1, a TR-FRET signal is generated. The interaction of unlabelled agonists can then be studied in a competitive setting which shows a decrease in signal as soon as the interaction of the unlabelled partner exceeds that of CELT-335. Figure 3 shows that competition was similar for ∆9-THC (pKi 7.2 ± 0.6) and ∆9-THCV (pKi 7.0 ± 0.3), whereas the affinity for ∆9-THCA was considerably lower (pKi 5.8 ± 0.6).

How microplate readers support protein-protein interaction studies

Since such applications are often performed in high throughput, the conversion of a benchtop assay into an automated high-throughput screening (HTS) assay comes with specific limitations. An important element here is the sensitivity and speed of the microplate reader, as well as its automation compatibility. By analyzing the binding curves of interaction measurements, valuable insights into the binding kinetics, dissociation constants, and stoichiometry of PPIs can be obtained. Many readers including all BMG LABTECH devices, also offer kinetic measurements supporting the measurement of binding curves.

® FSX” width=”300″ height=”300″ /> Figure 4. BMG LABTECH´s PHERAstar® FSX is an HTS-dedicated multi-mode microplate reader. It is equipped with dedicated features that facilitate the study of protein-protein interactions.

HTS-dedicated multi-mode microplate readers like BMG LABTECH´s PHERAstar® FSX (Figure 4), are the ideal measurement platform to address these points. They provide full flexibility in assay choice due to the availability of all commonly used detection modes combined with the performance required in screening campaigns. Beyond this, the PHERAstar® FSX plate reader can be equipped with dedicated features that can be used to analyze protein interactions with even greater detail and sensitivity. Features such as on-board reagent injectors, the simultaneous detection of two emission signals (SDE) and ultra-fast sampling rate enable to kinetically analyze interactions in real time in high throughput. Furthermore, compatibility with various robotics platforms and software interfaces allow full measurement automatization.



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If you are interested in further details about microplate-based evaluation of protein-protein interaction, please contact us at [email protected].

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