Abstract
Protein–protein interactions (PPIs) are an essential part of correct cellular functionality, making them increasingly interesting drug targets. While Förster resonance energy transfer-based methods have traditionally been widely used for PPI studies, label-free techniques have recently drawn significant attention. These methods are ideal for studying PPIs, most importantly as there is no need for labeling of either interaction partner, reducing potential interferences and overall costs. Already, several different label-free methods are available, such as differential scanning calorimetry and surface plasmon resonance, but these biophysical methods suffer from low to medium throughput, which reduces suitability for high-throughput screening (HTS) of PPI inhibitors. Differential scanning fluorimetry, utilizing external fluorescent probes, is an HTS compatible technique, but high protein concentration is needed for experiments. To improve the current concepts, we have developed a method based on time-resolved luminescence, enabling PPI monitoring even at low nanomolar protein concentrations. This method, called the protein probe technique, is based on a peptide conjugated with Eu3+ chelate, and it has already been applied to monitor protein structural changes and small molecule interactions at elevated temperatures. Here, the applicability of the protein probe technique was demonstrated by monitoring single-protein pairing and multiprotein complexes at room and elevated temperatures. The concept functionality was proven by using both artificial and multiple natural protein pairs, such as KRAS and eIF4A together with their binding partners, and C-reactive protein in a complex with its antibody.
Highlights
PPIs, most importantly as there is no need for labeling of either interaction partner, reducing potential interferences and overall costs
PPIs have been regarded as difficult drug targets, mostly because the interacting regions of PPIs are often flat and shallow, whereas small molecules often prefer binding to well-defined pockets, as in the case of enzymes and G-protein-coupled receptors, the most frequent drug targets today
Interactions between proteins are a fundamental part of correct cellular functionality, and impaired interactions may lead to various disease states
Summary
Interactions between proteins are a fundamental part of correct cellular functionality, and impaired interactions may lead to various disease states. EIF4H and PDCD4 gave only a modest signal when individual proteins were measured When they were assayed in complex with eIF4A, a clear TRL signal increase was detected at elevated temperatures (Figure 4). Many interactions have been reported to produce a significant stabilizing effect in complex with a binding partner when compared to individual proteins, and the strategy has been successfully applied especially in PLI inhibitor screening.[41,42] We did not observe any major thermal shift with eIF4A and its binding partners, and we chose to investigate KRAS, which is reported to be responsive to thermal stabilization.[43] As a KRAS interaction partner, we selected a guanosine diphosphate (GDP)-KRAS-specific designed ankyrin repeat protein (DARPin), K27, which is known to have inhibitory properties on KRAS activation and interactions with other proteins.[44] First, to prove the specificity and to estimate the binding affinity of K27 for KRAS, a quenching resonance energy transfer nucleotide exchange assay was performed using 50 nM KRAS.[26,45,46] Based on these results, IC50 values close to the KRAS concentration were calculated for K27 and for GDP acting as a control (Figure S8). The functionality in the microtiter plate format provides a good starting point for these assays and for further studies to understand the mechanism behind the method to improve its function
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
