Multiply labeling proteins for studies of folding and stability

Fluorescence spectroscopy is a powerful method for monitoring protein folding in real-time with high resolution and sensitivity, but requires the site-specific introduction of labels into the protein. The ability to genetically incorporate unnatural amino acids (Uaas) allows for the efficient synthesis of fluorescently labeled proteins with minimally perturbing fluorophores. Here, we describe recent uses of labeled proteins in dynamic structure determination experiments and advances in unnatural amino acid incorporation for dual site-specific fluorescent labeling. The advent of increasingly sophisticated bioorthogonal chemistry reactions and the diversity of Uaas available for incorporation will greatly enable protein folding and stability studies.