Binding Affinity Quantifications of the Bacteriophage Mu DNA Modification Protein Mom Using Microscale Thermophoresis (MST)

Shubha Udupa; Valakunja Nagaraja; Shweta Karambelkar

Binding Affinity Quantifications of the Bacteriophage Mu DNA Modification Protein Mom Using Microscale Thermophoresis (MST)

2022

Shubha Udupa | Valakunja Nagaraja | Shweta Karambelkar

Epigenetic modifications play diverse roles in biological systems. Nucleic acid modifications control gene expression, protein synthesis, and sensitivity to nucleic acid-cleaving enzymes. However, the mechanisms underlying the biosynthesis of nucleic acid modifications can be challenging to identify. Studying protein-ligand interactions helps decipher biosynthetic and regulatory pathways underlying biological reactions. Here, we describe a fluorescence labeling-based quantitative method for unraveling the biomolecular interactions of bacteriophage Mu DNA modification protein Mom with its ligands, using microscale thermophoresis (MST). Compared to traditional methods for studying protein-biomolecular interactions, MST requires significantly lower sample amounts, volumes, and analysis time, thus allowing screening of a large number of candidates for interaction with a protein of interest. Another distinguishing feature of the method is that it obviates the need for protein purification, often a time- and resource-consuming step, and works well with whole or partially purified cell extracts. Importantly, the method is sensitive over a broad range of molecular affinities while offering great specificity and can be used to interrogate ligands ranging from metal ions to macromolecules. Although we established this method for a DNA modification protein, it can easily be adapted to study a variety of molecular interactions engaged by proteins.

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