Designing Caspase-3 Sensors for Imaging of Apoptosis in Living Cells

• Type of Material: Article
• Authors: Ning Chen, Georgia State University; Yun Huang, Georgia State University; Lily Yang, Emory University; Rihe Liu, University of North Carolina; Jenny J. Yang, Georgia State University
• Language: English
• Date: 2009-01-01
• Publisher: Wiley: 12 months
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim
• Final Published Version (URL): http://dx.doi.org/10.1002/chem.200901439
• Title of Journal or Parent Work: Chemistry - A European Journal
• ISSN: 0947-6539
• Volume: 15
• Issue: 37
• Start Page: 9311
• End Page: 9314
• Grant/Funding Information: This work is supported in part by the following sponsors: GM-70555 to J.J.Y.; GSU Molecular Basis of Disease Predoctoral Fellowships to N.C.
• Keywords: apoptosis; ACTIVATION; MECHANISMS; Science & Technology; Chemistry; DEATH; enhanced green fluorescent proteins; PROTEOLYSIS; caspase-3; GREEN FLUORESCENT PROTEIN; protease sensors; Physical Sciences; ratiometric measurements; Chemistry, Multidisciplinary
• Research Categories: Health Sciences, Pharmacy; Chemistry, General

Abstract

A sensitive and specific caspase-3 sensor, based on a single enhanced green fluorescent protein (EGFP) to avoid the cross-talk from the overlap of cyan fluorescent protein (CFP) excitation or yellow fluorescent protein (YFP) emission spectra as observed in most FRET probes, was reported. A ratiometric protease sensor was designed by grafting a caspase-3-specific cleavage linker at a sensitive loop location (Glu172) relative to the chromophore, taking advantage of the EGFP's high resistance to proteases. To obtain optimal signal change and kinetic properties, two helical sequences were used to extend the caspase-3 cleavage linker and to improve enzymatic accessibility in solution. The comparison between dual cleavage sequence (DEVD) and a single sequence in caspase-3 sensors was also conducted. Moreover, to achieve enzymatic specificity, a recognition sequence (VDEVDG) with preferable residues from P5 to P1 was inserted into EGFP to create another sensor denoted as EGFP-C3B.