Differential scanning fluorimetry (DSF) is a practical and accessible technique that allows the assessment of multiphasic unfolding behavior resulting from subsaturating binding of ligands. Multiphasic unfolding is indicative of a heterogenous protein solution, which frequently interferes
with crystallization and complicates functional characterization of proteins of interest. Along with UV–Vis spectroscopy, DSF was used to guide purification and crystallization improvements for the pyridoxal 5′‐phosphate (PLP) dependent transaminase BioA from Mycobacterium
tuberculosis. The incompatibility of the primary amine‐containing buffer 2‐amino‐2‐(hydroxymethyl)‐1,3‐propanediol (Tris) and PLP was identified as a significant contributor to heterogeneity. It is likely that the utility of DSF for ligand‐binding
assessment is not limited to the cofactor PLP but will be applicable to a variety of ligand‐dependent enzymes.