The Molecular Detective’s Tool Kit: In Vivo Sensing by Fluorescent Protein FRET
Annalee Nguyen
Department of Chemical Engineering
UCSB Doctoral Candidate
Date:
Tuesday, January 17, 2006
Time: 4:00 p.m.
Place: Engineering II, Room 3361
ABSTRACT
A complete understanding of a biological system necessitates a thorough investigation of the functions and specificities of the intracellular components involved. Though in vitro methods continue to provide valuable information on the properties of proteins, the development of in vivo techniques would facilitate the collection of more detailed information of physiological or pathological relevance. This aim has driven the development of new tools and techniques suited for detection of biological activities in living samples. Fluorescent protein Förster resonance energy transfer (FRET) is an attractive choice for probing intracellular events, but the low efficiency of most available pairs limits the utility of the technique. To address this problem, a cyan-yellow fluorescent protein pair was optimized for FRET using directed evolution. The best pair of variants, CyPet-YPet, exhibited a seven-fold improvement in dynamic range over the parental pair when measured in vitro. The improved utility of CyPet and YPet enabled flow cytometric sorting on the basis of fluorescent protein FRET for the first time. To facilitate enzyme evolution studies, cells expressing an active proteolytic enzyme were isolated from inactive background present in 105-fold excess using the FRET signal change as an indicator of enzyme function. In addition, a FRET-based protein-protein interaction screen was developed, and enabled identification of peptide ligands to a given target protein in living cells. These tools and techniques developed for fluorescent protein FRET applications create opportunities for expansion of the utility of FRET in the discovery and study of biological interactions and activities.