A Systematic Approach for the Purification of Fluorophore‐Labeled Proteins via Anion Exchange Chromatography

Förster resonance energy transfer (FRET), an optical distance ruler, provides the unique ability to monitor molecular interactions and conformational changes in single biomacromolecules and multi-subunit complexes. It has proven to be a powerful tool to study enzymatic reactions, membrane transport, protein folding, but also the properties of nucleic acids, proteins, molecular motors, and many other biological systems and processes. A prerequisite for FRET is targeted (covalent) labeling of macromolecules with two distinct fluorophores. Here, we present a strategy for stochastic labeling of protein residues with the required donor and acceptor dyes via anion exchange chromatography. While this technique has been used before for this purpose, we provide a conceptual basis to systematically design a purification protocol for an arbitrary choice of fluorophores. By characterizing the interaction of hydrolyzed fluorophore-maleimides with the column material, we are able to select (and predict) which pairs of fluorophores allow a successful purification of donor-acceptor-labeled protein with yields up to 98%. We demonstrate the capabilities of the method for bulk and single-molecule FRET assays of various bacterial substrate-binding proteins, which are examples of soluble, folded, and well-characterized protein systems.