The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention.Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed.First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.
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