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Microscopy Reference Library

FRET Biosensor Fluorescent Proteins

Aside from their utility as fusion partners to report on protein localization in multiple colors, fluorescent proteins are also useful as reporters of gene activation, markers to follow cell lineage during development, monitors of protein dynamics, transgenic probes, tracking virus particle localization, protein complementation assays, and chromophore assisted light inactivation (CALI), among a host of other applications. Fluorescent proteins have also been cleverly used to create highly specific biosensors to monitor a wide spectrum of physiological processes, including pH fluctuations, calcium wave induction, cyclic nucleotide messenger effects, membrane potential differences, signaling, phosphorylation, redox reactions, and apoptosis.

Lalonde, S., Ehrhardt, D. W., and Frommer, W. B.

Shining light on signaling and metabolic networks by genetically encoded biosensors.  Current Opinion in Plant Biology 8: 574-581 (2005).  A comprehensive review article on fluorescent protein biosensors covering calcium transients, pH, halides, cyclic AMP, protein kinases, GTPases, glucose metabolism, apoptosis, redox potential, and lipids. Also discussed are basic concepts in how these FRET sensors are developed.

Looger, L. L., Lalonde, S., and Frommer, W. B.

Genetically encoded FRET sensors for visualizing metabolites with subcellular resolution in living cells.  Plant Physiology 138: 555-557 (2005).  The authors present a brief overview of the challenges presented in constructing fluorescent protein biosensors. Review are signal-to-noise, sensitivity, calibration, temporal and spatial resolution, and multiplexing.

Mank, M. and Griesbeck, O.

Genetically encoded calcium indicators.  Chemical Reviews 108: 1550-1564 (2008).  Oliver Griesbeck presents a detailed review of genetically encoded FRET-based calcium biosensors. In addition to examining studies on kinetics and the comparisons and contrasts between in vivo and in vitro performance, the use of these probes for detecting neuronal activity is described.

Palmer, A. E. and Tsien, R. Y.

Measuring calcium signaling using genetically targetable fluorescent indicators.  Nature Protocols 1: 1057-1065 (2006).  An excellent "how to" review on the basic aspects of using fluorescent protein FRET biosensors for observing calcium transients. The authors provide detailed step-by-step instructions for preparing the reagents as well as configuring instrument (microscope) parameters. Highly recommended for beginners.

Piljic, A. and Schultz, C.

Simultaneous recording of multiple cellular events by FRET.  ACS Chemical Biology 3: 156-160 (2008).  The authors describe the use of multiple ratiometric FRET sensors in parallel to detect simultaneous events through spatial distributions and spectral signatures. Included are sensors for calmodulin-dependent kinase, a membrane-bound kinase, and a FRET probe based on annexin A4.

Selvin, P. R.

The renaissance of fluorescence resonance energy transfer.  Nature Structural Biology 7: 730-734 (2000).  Although published over a decade ago, this review by Paul Selvin provides a relevant and excellent overview of the application of fluorescent protein FRET biosensors for detecting events in living cells. The review includes a discussion of new optical methods for detecting FRET.

VanEngelenburg, S. B. and Palmer, A. E.

Fluorescent biosensors of protein function.  Current Opinion in Chemical Biology 12: 60-65 (2008).  Drs. Palmer and VanEngelenburg discuss the vast array of FRET biosensors available for examining functions in biological systems and present a compilation of techniques and sensor molecules. The review article contains numerous references to earlier literature on the subject.

Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M., and Miyawaki, A.

Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.  Proceedings of the National Academy of Sciences U.S.A. 101: 10554-10559 (2004).  An excellent demonstration of how FRET efficiency can be fine-tuned by introducing circularly permuted fluorescent proteins. The authors describe the construction of six new calcium sensors based on cyan and Venus variants with the latter being circularly permuted at several locations.

Campbell, R. E.

Fluorescent-protein-based biosensors: Modulation of energy transfer as a design principle.  Analytical Chemistry 81: 5972-5979 (2009).  Dr. Campbell presents a comprehensive analysis of FRET biosensor design using fluorescent proteins. Addressed are basic design principles, evaluating distance parameters, effects of orientation, and maximizing Förster radius values.

Frommer, W. B., Davidson, M. W., and Campbell, R. E.

Genetically encoded biosensors based on engineered fluorescent proteins  Chemical Society Reviews 38: 2833-2841 (2009).  The authors describe the basic concepts of fluorescent proteins, including members of the color palette, and their application as passive and active FRET biosensors. Many of the mechanisms and design criteria are discussed, along with a comprehensive listing of biosensor constructs that have proven useful.