A Red Cy3-Based Biarsenical Fluorescent Probe Targeted to a Complementary Binding Peptide
Small-molecule biarsenical multiuse affinity probes (MAPs) FlAsH and ReAsH,1,2 in conjunction with complementary protein tags, are important new tools for analyzing cellular function through live-cell imaging,3,4 targeted protein inactivation,5 and the measurement of protein dynamics and binding.6 In addition, MAPs serve as affinity reagents for isolating intact protein complexes for complementary structural measurements.7 These first-generation MAPs bind to a tetracoordinate arsenic group (TAG) binding motif (i.e., CCXXCC or FlAsHTAG) genetically engineered onto a protein of interest. They are superior to other targeted labeling strategies (such as the Halo-tag, the SNAP tag, and fluorescent proteins) in that the small peptide tag does not disrupt protein protein interactions nor perturb the correct trafficking of tagged proteins.8,9 The conserved interatomic distance (*6 Å) between the two arsenic moieties in FlAsH and ReAsH complicates the selective labeling of multiple proteins with different reporters. To overcome these limitations, we have synthesized a new biarsenical MAP (i.e., AsCy3) based on Cy3, a member of the cyanine dye family, whose well-recognized brightness and photostability facilitate their utility in single-molecule measurements. The large interatomic distance between the two arsenics in AsCy3 (*14.5 Å) coupled with the identification of a complementary high-affinity binding sequence CCKAEAACC (Cy3TAG) permits the simultaneous application of both AsCy3 and FlAsH to selectively label their respective binding TAGs in different proteins. In addition, the fluorescence of FlAsH overlaps with the absorption of AsCy3, which can act as an acceptor of fluorescence resonance energy transfer (FRET) to allow ratiometric measurements of protein association.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1000649
- Report Number(s):
- PNNL-SA-53300; KP1102010
- Journal Information:
- Journal of the American Chemical Society, 129(28):8672-8673, Journal Name: Journal of the American Chemical Society, 129(28):8672-8673
- Country of Publication:
- United States
- Language:
- English
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