Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Tuesday Speaker Abstracts

Genetically Encoded Dark Acceptors for Use in FRET and pcFRET Applications Craig Don Paul 1 , Daouda Traore 1 , Anne Pettikiriarachichi 2 , Matthew Wilce 1 , Seth Olsen 4 , Csaba Kiss 3 , Matthew Perugini 5 , Toby Bell 1 , Rodney Devenish 1 , Andrew Bradbury 3 , Mark Prescott 1 . 1 Monash University, Melbourne, Australia, 3 Los Alamos National Laboratory, Los Alamos, NM, USA, 5 La Trobe University, Melbourne, Victoria, Australia. 2 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia, 4 University of Queensland, Brisbane, Queensland, Australia, Fluorescent proteins are available with optical properties that make them suitable for use as FRET donor/acceptor pairs. Many have been used to construct biosensors for monitoring cellular events. The use of a non-fluorescent or dark acceptor for FRET applications has a number of advantages, amongst which is facilitating the use of fluorescent probes to monitor additional parameters, otherwise precluded by the presence of emission from a fluorescent acceptor. Here we report on the properties and application of two novel genetically encoded dark acceptors useful for FRET experiments. The first protein called Phanta, can be used as a photochromic FRET (pcFRET) acceptor and is best suited to FPs with green emission such as EGFP. Phanta has a very low fluorescence quantum yield (ФF = 0.008) and can be reversibly, and repeatedly photoswitched between one of two absorbing states (495 nm and 395 nm) on sequential exposure to cyan or violet light. pcFRET can be readily estimated from donor emission images acquired with Phanta in each of the two photoswitched states. The second protein called Ultramarine is an intensely blue coloured non-fluorescent (ФF, 0.004) monomeric protein. The broad absorbance spectrum of Ultramarine makes it suitable as an acceptor for a number of different fluorescent donors including those with cyan, green, yellow or orange emissions. Fluorescence lifetime imaging is required to monitor FRET in live cells when using Ultramarine.

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