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

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.

Qubit Fluorometer (Invitrogen, excitation 485 nm, emission 590 nm) was used in protein concentration determination. All biochemical assays were performed in a 10 µL final volume using triplicate reactions and three individual experiments unless otherwise indicated.

Eu 3+ -probe and Protein-Probe spectral characterization and lifetime measurements
The excitation and emission spectra of 1 was performed using emission at 700 nm, and the emission spectra (at 630-850 nm) was measured using excitation at 618 nm. All spectra were measured using 1 nm step and 5 nm bandwidth.
The lifetimes of 50 nM Eu 3+ -probe, the Protein-Probe solution (50 nM Eu 3+ -probe, 3.5 µM HIDC), and the Protein-Probe solution with sample containing 1000 nM anti-CRP mAb and 200 nM CRP were measured in a quartz cuvette in 40 µl. The sample was prepared by incubating the mAb and CRP first at RT, then 3 min at 85 °C before addition to the Protein-Probe solution. Measurements were performed using 340 nm excitation and 615 nm emission, delay and gate times were 0.1 ms, and the total measurement time was 3 ms.

Protein production and purification
Eukaryotic initiation factor 4A (eIF4A), eukaryotic translation initiation factor (eIF4H), and programmed and MT2 (2.2 µM) in 10 µL 3 . Concentration of an unknown bio-BSA was determined similarly and compared to the biotin standard. The TRL-signals were monitored after 5 min incubation at RT.

Proof-of-concept streptavidin/bio-BSA assay
To observe SA binding to bio-BSA, assay using 20 nM BSA or bio-BSA was performed with 0-600 nM SA in Assay Buffer (10 mM HEPES, pH 7.5, 0.001 % (v/v) Triton X-100). After 5 min incubation, 65 µL of the Protein-Probe (7.7 mM Na2HPO4, 6.1 mM citric acid, pH 4, 0.01% Triton X-100, supplemented with 6 µM HIDC, and 4.5 nM Eu 3+ -probe), was added on top of 8 µL of protein-protein interaction (PPI) reaction. TRLsignals were monitored after 5 min incubation at room temperature (RT). The SA/bio-BSA interaction was confirmed in biotin titration, using 20 nM BSA or bio-BSA in the presence of 200 nM SA and 0-10 µM biotin. PPI was monitored using the Protein-Probe and TRL-signals were monitored as above.

Antibody-antigen interaction at RT and elevated temperatures
S-6 Anti-CRP and non-specific mAb assays were performed in Assay Buffer. For detection, the Protein-Probe contained 3.5 µM HIDC and 1 nM Eu 3+ -probe. To monitor CRP interaction with mAb, 0-100 nM CRP was combined with 0-500 nM anti-CRP mAb or two non-specific mAbs (hemoglobin or Lp-PLA2 mAb). After 10 min incubation at RT, the Protein-Probe was added in 65 µL on top of the 8 µL PPI reaction, and TRL-signals were monitored after 5 min.
For measurements in elevated temperatures, the thermal stability of the anti-CRP mAb (0.5-120 nM) was monitored using thermal ramping from 50 to 95 °C and 5 °C increments. All samples were prepared at RT and thereafter incubated for 3 min at each temperature before the Protein-Probe was added. TRL-signals were monitored after 5 min of incubation at RT. The CRP/mAb interaction was monitored using the same assay protocol as with the mAb alone, by combining 0-50 nM CRP with 2-30 nM anti-CRP mAb. TRL-signals were monitored as above. The same protocol was also used to monitor interaction with non-specific antihemoglobin mAb (10 nM) with 5 or 10 nM CRP.

eIF4A interaction control, thermal stability, and interactions measured at elevated temperatures
Assay conditions. All biochemical assays were performed in a 10 µL final volume using triplicate reactions methods. In Protein-Probe assays, PDCD4 was monitored using a protein concentration of 150 and 300 nM and eIF4H at 500 and 1000 nM concentrations. In SYPRO Orange assays the concentrations were 2 µM for PDCD4 and 2 or 6 µM for eIF4H. Samples were incubated with heating for 3 min (25-70 °C using 5 °C increments) before Protein-Probe addition, followed by TRL-signal monitoring after 5 min. SYPRO Orange assays were performed in a single step using 5x SYPRO Orange directly in heating. For further assays with the Protein-Probe, we selected PPI buffer without Triton X-100 supplement. Interaction of eIF4A (75 nM) with PDCD4 (0-300 nM) or eIF4H (0.5 or 1 µM), and interaction of 0.5 µM eIF4H with 150 nM PDCD4 were observed. The samples were incubated for 3 min at 45-75 °C using 5 °C increments, after which the Protein-Probe was added. TRL-signals were monitored after 5 min as described previously. 25-95 °C and TRL-signals were monitored using 5 °C increments. SYPRO Orange assay was monitored at the same temperatures but using single-step protocol. The interaction between 50 nM GMPPNP-KRAS or GDP-KRAS and 100 nM K27 was monitored in thermal ramping using the Protein-Probe. Samples were incubated and monitored as above from 25 to 95 °C.

Data analysis
The S/B ratio was calculated as µmax/µmin, specific signal as µmax-µmin, and coefficient variation (CV%) as (σ/µ) x 100. In these formulas, µ is the mean value and σ is the standard deviation (SD). The data were