linear approaches to intramolecular f？rster resonance energy transfer probe measurements for quantitative modeling文档文档.pdf

¨ Linear Approaches to Intramolecular Forster Resonance Energy Transfer Probe Measurements for Quantitative Modeling 1,2 1 1 3 Marc R. Birtwistle *, Alexander von Kriegsheim , Katarzyna Kida , Juliane P. Schwarz , Kurt I. Anderson3, Walter Kolch1 1 Systems Biology Ireland, University College Dublin, Belfield, Republic of Ireland, 2 Cancer Research Center, Georgia Health Sciences University, Augusta, Georgia, United States of America, 3 Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom Abstract ¨ Numerous unimolecular, genetically-encoded Forster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find tha