Figure 1

Strategies for site-specific labeling of proteins to detect folding and stability in vivo. (A) The autofluorescent green protein is appended to the protein of interest (here maltose-binding protein) and the latter dictates the spectroscopic behavior of the protein-reporter tag. The same fusion scheme applies for enzymes (e.g., CAT) whose activity serves as readout of the analysis. (B) Complementation assay. The target protein (here maltose-binding protein) is sandwiched between two reporter proteins or between the two halves of one reporter. Efficient folding brings both parts of the reporter unit into sufficient proximity to generate a readout signal. (C) Small-molecule labeling using peptide tags. The peptide tag can be introduced in the middle of the protein sequence (here cellular retinoic acid binding protein with a tetracysteine motif highlighted in yellow and FlAsH ligated to it) and the intensity of the emitted fluorescent signal can be used to determine conformationally distinct populations, provided the specific signatures of the folded and unfolded states are pre-determined for each experiment.