Application Notes

1 Protein-Protein Interaction Analysis in Different Buffer Systems Application Note NT-MO-012 Using MST to analyse the binding of the β-Lactamase TEM1 to BLIP Moran Jerabek-Willemsen 1 & Gideon Schreiber 2 1) NanoTemper Technologies GmbH, Munich, Germany 2) Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel Abstract The analysis of protein-protein interactions plays an important role in understanding many biological processes. MicroScale Thermophoresis (MST) permits a robust and fast analysis of protein- protein interactions even in highly complex biological liquids. Here we have investigated the binding of TEM1 β – Lactamase to BLIP (β – Lactamase Inhibitory Protein) using MicroScale Thermophoresis. The experiments were performed as in 50 % mammalian cell lysate, as well as in standard buffer. The TEM1-BLIP system is very well characterized by various biophysical methods, such as Surface Plasmon Resonance (SPR). The role of residue substitutions at the interface of the proteins was easily studied using TEM1 and BLIP mutants. Dissociation constants (K d ) determined for TEM1 and BLIP show an excellent agreement with previously published data. Introduction Protein–protein interactions play an essential role in many biological processes. Specific non-covalent interactions stabilize the structure of macromolecular assemblies. Fig. 1: Structural representation of the TEM1-BLIP complex. TEM1 is represented in cyan. BLIP is shown in white. TEM1 contact residues are in yellow. BLIP mutants with decreased affinity for TEM1 are represented in red, and the mutant W150A and W112A residues of BLIP are identified by a black arrow. The Image is slightly modified from Wang et al., 2007. The binding of one protein to another involves many contact residues and a relatively large interface that is rather planar. Flexibility of contact residues, chemical and physical properties, as well as solvent molecules

• Cover