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Proceedings Paper

Fluorescence-based characterization of genetically encoded peptides that fold in live cells: progress toward a generic hairpin scaffold
Author(s): Zihao Cheng; Robert E. Campbell
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Paper Abstract

Binding proteins suitable for expression and high affinity molecular recognition in the cytoplasm or nucleus of live cells have numerous applications in the biological sciences. In an effort to add a new minimal motif to the growing repertoire of validated non-immunoglobulin binding proteins, we have undertaken the development of a generic protein scaffold based on a single &bgr;-hairpin that can fold efficiently in the cytoplasm. We have developed a method, based on the measurement of fluorescence resonance energy transfer (FRET) between a genetically fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), that allows the structural stability of recombinant &bgr;-hairpin peptides to be rapidly assessed both in vitro and in vivo. We have previously reported the validation of this method when applied to a 16mer tryptophan zipper &bgr;-hairpin. We now describe the use of this method to evaluate the potential of a designed 20mer &bgr;-hairpin peptide with a 3rd Trp/Trp cross-strand pair to function as a generic protein scaffold. Quantitative analysis of the FRET efficiency, resistance to proteolysis (assayed by loss of FRET), and circular dichroism spectra revealed that the 20mer peptide is significantly more tolerant of destabilizing mutations than the 16mer peptide. Furthermore, we experimentally demonstrate that the in vitro determined &bgr;-hairpin stabilities are well correlated with in vivo &bgr;-hairpin stabilities as determined by FRET measurements of colonies of live bacteria expressing the recombinant peptides flanked by CFP and YFP. Finally, we report on our progress to develop highly folded 24mer and 28mer &bgr;-hairpin peptides through the use of fluorescence-based library screening.

Paper Details

Date Published: 14 February 2007
PDF: 10 pages
Proc. SPIE 6449, Genetically Engineered and Optical Probes for Biomedical Applications IV, 64490S (14 February 2007); doi: 10.1117/12.717274
Show Author Affiliations
Zihao Cheng, Univ. of Alberta (Canada)
Robert E. Campbell, Univ. of Alberta (Canada)

Published in SPIE Proceedings Vol. 6449:
Genetically Engineered and Optical Probes for Biomedical Applications IV
Samuel Achilefu; Alexander Pavlovich Savitsky; Rebekka M. Wachter; Darryl J. Bornhop; Ramesh Raghavachari, Editor(s)

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