Nanomolar Protein–Protein Interaction Monitoring with a Label-Free Protein-Probe Technique

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 Eu 3+ 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.

Sensitive, homogeneous, and label-free protein-probe assay for antibody aggregation and thermal stability studies

“…Previously, we demonstrated the Eu 3+ -probe for monitoring protein stability and interactions and protease activity. 35 , 36 , 41 Here, we further developed the Protein-Probe method to detect mAb aggregates.…”

“…Based on our previous findings related to protein-protein interactions, we assumed that these differences could be caused by varying levels of mAb aggregation in the samples. 36 The Protein-Probe method has a proven high sensitivity in the detection of protein denaturation, and we assumed it can also potentially monitor early aggregation. Our monitoring of six temperature-stressed mAbs at RT with the Protein-Probe indicated that these mAbs had different stability and susceptibility to aggregation ( Figure 3a ).…”

“…Previously, we demonstrated the Eu 3+ -probe for monitoring protein stability and interactions and protease activity. 35,36,41 Here, we further developed the Protein-Probe method to detect mAb aggregates. The Protein-Probe method is based on measuring the TRLsignal of the Eu 3+ -probe, consisting of a protein-binding peptide probe and a conjugated Eu 3+ -chelate (Figure 1a-c).…”

“…In addition, our Protein-Probe method requires only a fraction of the sample compared to other introduced methods, and it is also suitable for protein stability monitoring, giving additional information when assayed with thermal ramping. 35 , 36 Here, we used the Protein-Probe in a proof-of-principle study to monitor a panel of monoclonal antibodies (mAbs) and their heat- or pH-induced aggregation (RT measurements), and also stability in thermal ramping. Both monitored parameters – aggregation behavior and thermal stability – can give important information on therapeutic mAbs.…”

Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins

“…The Eu-probe was purified as described before, [31] and the concentration was determined using the DELFIA technique and a commercial EuCl 3 standard from PerkinElmer Life and Analytical Sciences, Wallac (Turku, Finland). The protein substrates, recombinant pertussis toxin (PTX), G protein alpha I subunit (Gαi) [33], human Ras GTPase-activating protein 1 (p120GAP) [34], wild type Kirsten RAt Sarcoma virus (KRAS, 2-188), human son of sevenless SOS1 catalytic domain (SOS cat , 564-1048) [35], and eukaryotic initiation factor 4A1 (eIF4A1) [32] were kind gifts from our collaborators. Malate dehydrogenase (MDH) was purchased from Roche (Basel, Switzerland).…”