In many biological processes, interactions between different proteins play a major role. For example, the structure of macromolecular assemblies is stabilized by certain non-covalent interactions. Many contact residues and a large planar interface are involved during the binding of one protein to another.
The binding can be affected by the flexibility of physical and chemical properties, contact residues, and solvent molecules contained in the interface. It should be noted that only a small amount of the contact residues is involved in the free energy of binding, as examined by Alanine-scanning Mutagenesis (Wang et al., 2007, Reichmann et al., 2005).
In order to understand macromolecular interactions in biological systems, a fast, reliable technique is required to investigate binding affinities. This article demonstrates an example of such an analysis using MicroScale Thermophoresis (MST) technology.
TEM1 is a β–Lactamase enzyme that offers resistance against β-Lactam antibiotics. These antibiotics are extensively used as antimicrobial antibiotics. The active peptide bound to the β- Lactam ring is selectively cleaved by the β–Lactamase enzyme.
β-Lactamase Inhibitory Protein (BLIP), which is a 154-amino acid protein expressed by the soil bacterium Streptomyces clavuligerus, inhibits TEM1 - β-Lactamase (Albeck and Schreiber, 1999).
In this experiment, the researchers examined the results of well-defined mutations of BLIP and TEM1 residues on the dissociation constant (Kd) both in mammalian 293T cell lysate and standard buffer. For this study, a combination of fluorescently-labeled TEM1 (NT-647 dye) and a fluorescent-fusion protein - Ypet-BLIP was used.
MST technology is suitable for studying protein-protein interactions in solution, as shown by the above experiments. The established binding affinities agree well with those measured on the ProteOn XPR36 system (Table 1).
Buffer independency is a major benefit of the MST Technology. MST is also a perfect tool for studying binding interactions in highly complex biological specimens such as blood serum and cell lysates.
As interactions occur in biological fluids, the same should be measured under similar experimental conditions. The application of fluorescence-fusion proteins in MST experiments (Figure 5) presents new avenues to study binding affinities under in vivo-like conditions. This can provide a better understanding of macromolecular interactions occurring in crowded environments.
Figure 5. Binding of Ypet-BLIP to TEM1 protein analyzed in 50% mammalian cell lysate. TEM1 was titrated in 1:1 dilutions in MST-buffer. Then Ypet-BLIP was added into 200µl of 293T cell lysate to a final concentration of 20nM. 10µl of BLIP-containing cell lysate was mixed with 10 µl TEM1 dilutions. For measurements, samples were filled into standard treated capillaries (n = 3). A Kd of 5.7nM ± 0.75nM was determined for this interaction.
Produced from materials originally authored by Moran Jerabek-Wilemsen1 and Gideon Schreiber2 from:
- NanoTemper Technologies GmbH, Munich, Germany
- Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
- Wang et al., Thermodynamic investigation of the role of contact residues of beta -lactamase - inhibitory protein for binding to TEM -1 beta - lactamase. J Biol Chem. 2007 Jun 15;282(24):17676 -84.(2007).
- Reichmann et al., The modular architecture of protein -protein binding interfaces. Proc Natl Acad Sci U S A. Jan 4;102(1):57 -62 - (2005)
- Jerabek -Willemsen et al., Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol. Aug;9(4):342 -53 (2011).
- Wienken et al., Protein -binding assays in biological liquids using microscale thermophoresis. Nat Commun. Oct 19;1:100 (2010).
- Albeck S and Schreiber G, Biophysical characterization of the interaction of the b - lactamase TEM -1 with its protein inhibitor BLIP, Biochemistry 38, 11 –21 (1999).
- Philipp et al., Protein -binding dynamics imaged in a living cell . Proc Natl Acad Sci U S A. Jan 31;109(5):1461 -6 (2012).
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MicroScale Thermophoresis (MST) technology is a unique method to study binding affinities in an easy, fast and precise way. Four Monolith Series instruments were introduced since 2010.
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