Background

[Background] Biological background: Biological entities have evolved a plethora of epigenetic modifications in their nucleic acids, ranging from simple methylation to complex moieties derived from sugars, amino acids, polyamines, and other cellular metabolites (Boccaletto et al., 2018; Sood et al., 2019). Epigenetic modifications regulate gene expression, mRNA translation, and cellular stress responses, and protect bacteriophages from DNA-damaging immune systems of bacterial hosts (Kahmann, 1983; Hattman, 1999; Choi et al., 2014; Gu et al., 2014; Reynolds et al., 2014; Bryson et al., 2015; Hutinet et al., 2019). The ability to target nucleic acid modifications has broad applications in designing antibiotics and antifungal strategies through the impairment of modification pathways unique and essential to pathogens (Koh and Sarin, 2018). Similarly, the fields of phage therapy and microbiome engineering stand to benefit from the discovery of DNA modification pathways that can protect the DNA payload from a wide range of host immune systems. Novel DNA modifications are constantly being discovered in phages, presenting new puzzles surrounding their biosynthesis and function (Lee and Weigele, 2021; Lee et al., 2022). Studying molecular interactions opens a window into the inner workings of macromolecules, cells, and underlying mechanistic and regulatory pathways (Corbeski et al., 2018; Karambelkar et al., 2020). Here, we describe interaction studies of the DNA modification protein Mom in the context of its metal ion and small molecule cofactor interactions (Karambelkar et al., 2020) using the technique of microscale thermophoresis (MST).

Technical background: MST offers a simple method to quantitatively investigate protein-ligand interactions, and is based on thermophoresis, the movement of molecules across thermal gradients (Jerabek-Willemsen et al., 2011). Ligand-induced changes in size, charge, hydration shell, or conformation of a macromolecule alter its thermophoretic movement (Jerabek-Willemsen et al., 2011). Using a titration approach, MST enables the measurement of affinity constants of a wide variety of interactions in the binding equilibrium (Karambelkar et al., 2020; Osuna et al., 2020a, 2020b). MST requires extremely small sample amounts compared to conventional methods like isothermal calorimetry (ITC) or surface plasmon resonance (SPR), obviates ligand immobilization, and works over a broad range of binding affinities.

Another notable strength of MST over conventional approaches is its compatibility with complex mixtures and cell extracts (Lippok et al., 2012; Khavrutskii et al., 2013; Bartoschik et al., 2018). The ability to fluorescently tag a specific his-tagged protein of interest in a complex mixture of proteins, as highlighted in this protocol, not only obviates protein purification, often a rate limiting and cumbersome step, but also provides high sensitivity for detecting interactions in a near-native environment (Bartoschik et al., 2018).

Although MST measurements can be performed using intrinsic fluorescence of proteins (Seidel et al., 2012), labeling of the target proteins with a suitable fluorophore is required when using complex samples (Bartoschik et al., 2018). In routine labeling techniques for MST, fluorophores are covalently attached to lysine residues using NHS- or to cysteine residues using maleimide chemistry. However, these labeling methods require purified proteins and cannot be applied to a mixture of proteins. Moreover, it is not possible to predict where the fluorophore will bind to the protein—inhomogeneous protein-dye conjugates might even display destabilization, loss of functionality, or steric hindrance at the ligand binding site (Lindhoud et al., 2012). The above limitations are overcome by the selective and site-specific labeling of a protein of interest in a complex mixture. This can be achieved in various ways, such as by expressing in vivo a fusion of the protein of interest with a fluorescent protein like GFP (Khavrutskii et al., 2013; Magnez et al., 2017; Gao et al., 2021). However, such relatively large tags are not always desired for quantitative interaction analysis and also require the cloning of a suitable linker and fluorescence tag. Various other site-specific labeling strategies have been demonstrated, such as co-translational introduction of unnatural or modified amino acids, or labeling via specific amino acid sequences, including His-tags and tetracysteine motifs (Griffin et al., 1998; Krishnan et al., 2007; Nienberg et al., 2016). Among these, the His-tag is the most convenient, popular, and widely used affinity tag for purification, immobilization, or detection of proteins.

The tris-NTA/His-tag system comprises one of the smallest high-affinity recognition elements known to date (Braner et al., 2016). Fast, stoichiometric binding of tris-NTA conjugates enables in situ protein labeling of His-tagged proteins, making the system suitable for quantitative protein interaction analysis by MST (Lata et al., 2005). Because the tris-NTA/His-tag labeling is based on non-covalent coordinate bonds with the transition metal ion Ni(II), it is sensitive to reagents that compete or interfere with the labeling (Table 1).