Analysis of Major Royal Jelly Proteins Stability and Ligand Binding by Differential Scanning Fluorimetry

Royal jelly (RJ) is produced by the hypopharingeal and mandibular glands of bees. Up to 90% of the RJ proteins are the major royal jelly proteins (MRJPs) that exhibit biological properties. Protein stability is of interest for biotechnology and can be explored by using differential scanning fluorimetry (DSF). We tested the stability by using a fluorescence dye (SYPRO® Orange,1:1000 dilution) in the presence of different ligands for MRJP1 and MRJP2 at 2 µM concentration. The fluorescence intensities (FI) were measured using the Real-Time PCR System and were fitted as a function of temperature according to the Boltzmann equation to determine the melting temperature (Tm) at which the amount of folded and unfolded protein is equal. In general, ligands stabilize the protein when binding to it and increase the Tm. Due to the increased starting fluorescence of the oligomeric MRJP1, the Tm values were only determined for monomeric MRJP1 and MRJP2. ATP, histamine, and thiamine were shown to determine an increase of Tm for the monomeric MRJP1 in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl. For the ligand ATP, the minimum limit of binding starts at 1.5 mM concentration, for the ligand Histamine it starts at 0.6 mM concentration, while for the ligand Thiamine it starts at 3 mM concentration.