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Quantitative assessment of uorescent proteins
Paula J Cranfill1,2,5, Brittney R Sell1,5, Michelle A Baird1,5, John R Allen1,5, Zeno Lavagnino2,3,
H Martijn de Gruiter4, Gert-Jan Kremers4, Michael W Davidson1,6, Alessandro Ustione2,3 & David W Piston2,3
npg 201 6 Nature America, Inc. All rights reserved.
npg 201 6 Nature America, Inc. All rights reserved.
The advent of uorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized uorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autouorescent FPs is their tight b-barrel structure, which provides the rigidity and chemical environment needed for effectual uorescence. Despite the common structure, eachFP has unique properties. Thus, there is no single best FPfor every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is rightfor a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.
Live-cell fluorescence microscopy offers a powerful way to examine tissues, cells and subcellular components at functionally important scales of time and length1. The cloning of GFP from the jellyfish Aequorea victoria2 and its expression in non-jellyfish systems3 empowered fluorescence microscopy in cell biology and physiology. A major leap in FP technology came with the discovery of homologous FPs from corals and other anthozoans4.
Genetic manipulations performed on A. victoria GFP5 as well as non-jellyfish proteins6 have led to FPs covering spectral regions from blue to red with improved brightness and stability5,7,8.
Detailed discussions of FP mutations are found elsewhere1,5,8.
Some general properties of FPs are critical for understanding their function and utility. FPs are ~25 kD, which is large compared to organic fluorophores, and the entire protein structure appears to be essential to its fluorescence9. FP structures consist of 11 -strands surrounding a central -helix10, where the fluorophore arises from a few amino acids. During protein biogenesis, a reaction occurs among FP residues to form an extended conjugation5,
and fluorescence depends on rigidity and chemical environment within the protein structure11. Within the -barrel fold, mutations can change the local fluorophore environment and lead to variations in...