Abstract
Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides is thus an important task in systems biology, cell biology, biochemistry, and drug design. However, this task is often hampered by the drawbacks of classical biophysical methods for analysis of molecular interactions like surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC), which require immobilization of the interaction partners or very high sample concentrations. MicroScale Thermophoresis (MST) is an innovative method that offers the possibility to determine the important parameters of a molecular interaction, such as dissociation constant, stoichiometry, and thermodynamics. Moreover, it does so in a rapid and precise manner, with free choice of buffers or biological liquids, no need for sample immobilization, and very low sample consumption. Here we describe two MST assays in detail, which analyze (i) the interactions between certain peptide stretches of the eukaryotic RNA polymerase II and a protein subunit of the eukaryotic transcription elongation complex and (ii) interactions between N-terminal histone tail peptides and epigenetic reader proteins. These experiments show that MST is able to characterize protein-peptide interactions that are triggered by only minor changes in the peptide, for example, only one phosphorylation at a specific serine residue.