Microscale Thermophoresis as a Sensitive Method to Quantify Protein: Nucleic Acid Interactions in Solution
Microscale thermophoresis (MST) is a new method that enables the quantitative analysis of molecular interactions in solution at the microliter scale. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge, and hydration shell. Since at least one of these parameters is typically affected upon binding, the method can be used for the analysis of each kind of biomolecular interaction or modification of proteins or DNA. Quantitative binding parameters are obtained by using a serial dilution of the binding substrate. This section provides a detailed protocol describing the analysis of DNA–protein interactions, using the AT-hook peptides as a model system that bind to short double-stranded DNA.
Key wordsBinding assay Dissociation constant DNA–protein interactions Microscale thermophoresis Interaction affinity
The authors would like to thank Christoph J. Wienken the fruitful comments and suggestions for data analysis.
- 1.Reeves, R. and Nissen, M.S. (1990) The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure. J. Biol. Chem. 265, 8573–8582.Google Scholar
- 2.Aravind, L. and Landsman, D (1998) AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res. 26, 4413–4421.Google Scholar
- 3.Reeves, R. (2001) Molecular biology of HMGA proteins: hubs of nuclear function. Gene 277, 63–81.Google Scholar
- 4.Reeves, R. (2010) Nuclear functions of the HMG proteins. Biochim. Biophys. Acta 1799, 3–14.Google Scholar
- 5.Susbielle, G., et al. (2005) Target practice: aiming at satellite repeats with DNA minor groove binders. Curr. Med. Chem. Anticancer Agents 5, 409–420.Google Scholar
- 6.Strohner, R., et al. (2001) NoRC--a novel member of mammalian ISWI-containing chromatin remodeling machines. EMBO J. 20, 4892–4900.Google Scholar
- 7.Németh, A., et al. (2004) The chromatin remodeling complex NoRC and TTF-I cooperate in the regulation of the mammalian rRNA genes in vivo. Nucleic Acids Res. 32, 4091–4099.Google Scholar
- 8.Duhr, S. and Braun, D. (2006) Why molecules move along a temperature gradient. Proc. Natl. Acad. Sci. USA 103, 19678–19682.Google Scholar
- 9.Baaske, P., et al. (2007) Extreme accumulation of nucleotides in simulated hydrothermal pore systems. Proc. Natl. Acad. Sci. USA 104, 9346–9351.Google Scholar
- 10.Baaske, P., et al. (2010) Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew. Chem. Int. Ed. 49, 2238–2241.Google Scholar
- 12.Huth, J.R., et al. (1997) The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif. Nat. Struct. Biol. 4, 657–665.Google Scholar