Determination of affinities of lanthanide-binding proteins using chelator-buffered titrations

The recent discoveries of the first proteins that bind lanthanides as part of their biological function not only are relevant to the emerging field of lanthanide-dependent biology, but also hold promise to revolutionize the technologically critical rare earths industry. Although protocols to assess the thermodynamics of metal–protein interactions are well established for “traditional” metal ions in biology, the characterization of lanthanide-binding proteins presents a challenge to biochemists due to the lanthanides' Lewis acidity, propensity for hydrolysis, and high-affinity complexes with biological ligands. These properties necessitate the preparation of metal stock solutions with very low buffered “free” metal concentrations (e.g., femtomolar to nanomolar) for such determinations. Herein we describe several protocols to overcome these challenges. First, we present standardization methods for the preparation of chelator-buffered solutions of lanthanide ions with easily calculated free metal concentrations. We also describe how these solutions can be used in concert with analytical methods including UV–visible spectrophotometry, circular dichroism spectroscopy, Förster resonance energy transfer (FRET), and sensitized terbium luminescence, in order to accurately determine dissociation constants (Kds) of lanthanide–protein complexes. Finally, we highlight how application of these methods to lanthanide-binding proteins, such as lanmodulin, has yielded insights into selective recognition of lanthanides in biology. We anticipate that these protocols will facilitate discovery and characterization of additional native lanthanide-binding proteins, will motivate the understanding of their biological context, and will prompt their applications in biotechnology.