Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.Microscale thermophoresis (MST) is a biophysical technique that has seen increasing application in the study of biomolecular interactions thanks to its solution-based nature, its rapid application, modest sample demand, and the sensitivity of the thermophoresis effect to binding events. Here, we describe the use of MST in the study of chromatin interactions, with emphasis on the wide range of ways in which these experiments are set up and the diverse types of information they reveal. These aspects are illustrated with four very different systems: the sequence-dependent DNA compaction by architectural protein HMfB; the sequential binding of core histone complexes to histone chaperone APLF; the impact of the nucleosomal context on the recognition of histone modifications; and the binding of a LANA-derived peptide to nucleosome core. Special emphasis is given to the key steps in the design, execution, and analysis of MST experiments in the context of the provided examples.
Keywords: Binding affinity; DNA; HMf; Histones; MST; Nucleosome.