Competitive fluorescence polarization assay

Competitive fluorescence polarization measurement and data evaluation were performed as described by Simon and collaborators [62]. Four different fluorescent peptides were used f16E6 (fluorescein‐RTRRETQL), fRSK1 (fluorescein‐KLPSTTL), fpRSK1 (fluorescein‐KLPpSTTL), and MERS‐CoV. The MERS‐CoV peptide was labeled with sub‐stoichiometric FITC (Sigma‐Aldrich, St. Louis, MO, USA) in a basic HEPES buffer (pH 8.2), and the reaction was stopped with 100 mm TRIS. The peptide was buffer exchanged in order to remove fluorescent contaminants. Fluorescence polarization was measured with a PHERAstar (BMG Labtech, Offenburg, Germany) microplate reader by using 485 ± 20 and 528 ± 20 nm band‐pass filters (for excitation and emission, respectively). In direct FP measurements, a dilution series of the Ni‐ and Amylose‐affinity purified MBP‐PDZ proteins was prepared in a 20 mm HEPES pH 7.5 buffer that contained 150 mm NaCl, 0.5 mm TCEP, 0.01% Tween 20, and 50 nm fluorescently labeled peptide. In total, the polarization of the probe was measured at 8 different protein concentrations. In competitive FP measurements, the same buffer was supplemented with the protein to achieve a complex formation of 60–80%, based on the direct titration. Then, this mixture was used for creating a dilution series of the competitor (i.e., the studied peptides) and the measurement was carried out identically as in the direct experiment. Analysis of FP experiments was carried out using ProFit.