Engineered Protein-tag for Rapid Live-cell Fluorogenic Visualization of Proteins by Anionic Probes

An “OFF–ON–OFF” fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems

“…We also prepared a second PYP NQN mutant, by replacing the same three acidic residues with the neutral amino acids, Asn, Gln and Asn, since this mutant had been shown to react more rapidly with anionic PYPtag probes, as well as display better cellular expression levels. 41 Fluorescence analyses conrmed that the uorescence response of all three probes were quenched in their free state (Fig. 4 & S3 †), with each probe producing absorption maxima between 500-505 nm that were red shied in comparison to uorescein (absorption maxima of 491 nm) (Fig.…”

“…S16 †). 41 We then attempted to use the OFF-ON uorescence properties of a DNB probe to image intracellular reservoirs of PYPtag proteins present in the nuclei of cells. HEK293T cells transfected with genes encoding PYP 3R or PYP NQN fused with maltose-binding protein (MBP) and nuclear localization signal (NLS) were rst expressed for 24 h. Initial attempts to use F5-DNB2 to visualize MBP-PYP 3R -NLS and MBP-PYP NQN -NLS proteins in the nuclei of the transfected cells proved unsuccessful, because the F5-DNB2 probe was impermeable to the plasma membrane.…”

“…6, S18 & S24 †), which is likely due to greater cell expression levels for the PYP NQN -NLS constructs. 41 In vivo ON-OFF uorescent imaging of the proteolytic degradation of a short-lived PYP-tag labeled protein in living cells Real-time imaging of the proteolytic degradation of SLPs in cellular systems using an OFF-ON-OFF uorescence switch is a challenging goal, because the probe needs to rapidly label the SLP before signicant SLP degradation occurs. Furthermore, proteolytic hydrolysis of a uorescent probe-protein conjugate must produce a corresponding rapid turn 'OFF' of cellular uorescence.…”

“…Statistical analyses revealed that PYP NQN -NLS transfected cells emitted higher intensity fluorescence signals than PYP 3R -NLS transfected cells (Figures 6, S18 & S24), which is likely due to greater cell expression levels for the PYP NQN -NLS constructs. 41…”

Real-time imaging of cell-surface proteins with antibody-based fluorogenic probes

“…Due to the importance of cell-surface proteins in maintaining cell homeostasis as well as in dealing with pharmacological intervention, 1-3 methods for selectively detecting the target protein in native environments are in high demand to illustrate its function and evaluate the therapeutic outcomes. Methods relying on genetic engineering to fuse uorescent proteins directly or to introduce tags like SNAP or PYP proteins, 4,5 peptides, 6,7 or unnatural amino acids 8 followed by the secondary labeling of complementary reporters have been developed. Though valuable, these methods are not feasible to monitor the dynamics of endogenously expressed proteins and may suffer from overexpression issues.…”