Multiply labeling proteins for studies of folding and stability

Abstract

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 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 unnatural amino acids available for incorporation will greatly enable protein folding and stability studies.

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

“…Although high-purity site-specific dye-labeled nucleic acids are now commercially available, preparation of site-specifically labeled proteins at multiple residues is far more challenging. Advances in this field [reviewed in (108,109)] will allow the study of multiple reaction coordinates and coordinated motions in single subunit proteins. As an alternative [already suggested in 1999 (110)], smFRET can be combined with other single-molecule techniques not involving fluorescence.…”

Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection

“…5, 6 Fluorescence spectroscopy is a powerful tool for studying such processes, as it allows one to observe protein motions in real time under physiological conditions, including measurements in live cells. 7, 8 One can even obtain low resolution structural information using distance-dependent chromophore interactions such as Förster resonance energy transfer (FRET) and quenching by photo-induced electron transfer (eT). 9 To appropriately model protein motions, one needs a set of probes that are capable of accurately reporting on distance changes without disrupting the fold and function of the protein of interest.…”

Optical probes based on G protein‐coupled receptors – added work or added value?

“…As we discover new signalling modalities of GPCRs, like signalling from within the cell (Irannejad et al ., ; Lohse and Hofmann, ), we also face novel challenges for labelling strategies. Some interesting developments are seen by the incorporation of unnatural amino acids into proteins that offer unique chemistry for functional coupling in living cells and allow a site‐selective labelling in cells (Haney et al ., ). Such approaches allow the use of brighter organic fluorophores that were already used in vitro in single‐molecule FRET experiments (Tyagi and Lemke, ).…”