Application Notes

2 trapped in the interface, can affect the binding. Interestingly only a small fraction of the contact residues contribute significantly to the free energy of binding, as studied by Alanine-scanning Mutagenesis (Wang et al., 2007, Reichmann et al., 2005). A fast and reliable method to analyze binding affinities is important for understanding macromolecular interactions in biological systems. An example of such an analysis, using MicroScale Thermophoresis, is described in this application note. For more detailed information about MST Technology refer to Jerabek-Willemsen et al. 2011 and Wienken et al., 2010. β–Lactamase enzymes, such as TEM1, provide resistance to β-Lactam antibiotics, which are widely used as antimicrobial antibiotics. The enzyme can selectively cleave the active peptide bound in the β- Lactam ring. BLIP (β-Lactamase Inhibitory Protein) is a 154- amino acid protein, which is expressed by the soil bacterium Streptomyces clavuligerus. This protein can inhibit the TEM1 - β-Lactamase (Albeck and Schreiber, 1999). The TEM1-BLIP complex is represented in Fig. 1. In this study we have analyzed the consequences of well characterized mutations of TEM1 and BLIP residues on the dissociation constant (K d ) in standard buffer, as well as in mammalian 293T cell lysate. For this analysis, we have used fluorescently- labeled TEM1 (NT-647 dye), in addition to a fluorescent-fusion protein - Ypet-BLIP. For a detailed description of the Ypet-BLIP system refer to Philipp et al., 2011. Results In the first experiment, we investigated the binding of a NT-647-labeled TEM1 to WT BLIP or two other BLIP mutants, in which alanine was substituted for tryptophan at positions 112 and 150 of BLIP. The concentration of NT-647 labeled TEM1 was kept constant at 10 nM, while the concentration of BLIP was varied. After a short incubation, the samples were loaded into MST standard treated glass capillaries (K002, NanoTemper technologies GmbH, Germany) and MST analysis was performed. The calculated K d for the interaction between NT-647-labeled WT TEM1 and WT BLIP was 3.5 nM +/- 0.6 nM. We observed, as expected, approximately a 100-fold weaker binding affinity of NT647-labeled TEM1-WT to BLIP-W112A. A mutation of tryptophan to alanine at position 150 resulted in a 600-fold weaker K d , as compared to WT BLIP (Fig. 2). These results are in good agreement to previously published affinities determined by SPR (Table 1). Interestingly the signal observed in Figure 2C for binding of NT647 labeled TEM1-WT to BLIP-W150A, showed an opposite thermophoretic behavior as compared to TEM1 binding to BLIP-WT (Fig. 2A) and BLIP-W112A (Fig. 2B). Reichmann et al., 2005 and Wang et al., 2007 have described the anti-canonical nature of this mutation. Substitution of the tryptophan at position 150 of BLIP results in a strong conformational rearrangement, and significant change in the free energy of binding. Thermodynamic studies also indicate that this mutation leads to a significant loss of enthalpy. In addition, Wang et al. reported an increased number of water-bridged intermolecular hydrogen bonds in the complex of TEM-WT with BLIP-W150A. This contributes to a lower enthalpic driving force for binding and disrupts hydrophobic stacking interactions. Taken together, it is possible that the loss of enthalpy and change in the hydration shell is related to the different thermophoretic response.

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