TY - JOUR
T1 - A Fluorogenic AggTag Method Based on Halo- and SNAP-Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells
AU - Jung, Kwan Ho
AU - Kim, Sojung F.
AU - Liu, Yu
AU - Zhang, Xin
N1 - Funding Information:
We thank Dr. Gang Ning at the Penn State Microscopy Facility for acquisition of confocal and electron microscopy images. We thank Matthew Fares for providing purified Halo-tag and SNAP-tag proteins, Charles Wolstenholme for providing technical assistance in probe synthesis, and Leeann Grainger for providing plasmids encoding SOD1-SNAPf, Htt-Q110·Halo, and Htt-Q97·SNAPf. This work was supported by the Burroughs Wellcome Fund Career Award at the Scientific Interface (X.Z.), a Paul Berg Early Career Professorship (X.Z.), a Lloyd and Dottie Huck Early Career Award (X.Z.), and the Sloan Research Fellowship (X.Z.).
Publisher Copyright:
© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
PY - 2019/4/15
Y1 - 2019/4/15
N2 - Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1, based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe—P2, based on a SNAP-tag ligand bearing a green solvatochromic fluorophore—was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.
AB - Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1, based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe—P2, based on a SNAP-tag ligand bearing a green solvatochromic fluorophore—was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.
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U2 - 10.1002/cbic.201800782
DO - 10.1002/cbic.201800782
M3 - Article
C2 - 30609255
AN - SCOPUS:85062800825
SN - 1439-4227
VL - 20
SP - 1078
EP - 1087
JO - ChemBioChem
JF - ChemBioChem
IS - 8
ER -