Dimerization-dependent green and yellow fluorescent proteins

Abstract

Dimerization-dependent fluorescent proteins (ddFP) are a recently introduced class of genetically encoded reporters that can be used for the detection of protein interactions in live cells. The progenitor of this class of tools was a red fluorescent ddFP (ddRFP) derived from a homodimeric variant of Discosoma red fluorescent protein. Here, we describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants. These optimized variants offer several advantages relative to ddRFP including increased in vitro contrast and brightness for ddGFP and increased brightness and a lowered pK a for ddYFP. We demonstrate that both variants are useful as biosensors for protease activity in live cells. Using the ddGFP tool, we generated a highly effective indicator of endomembrane proximity that can be used to image the mitochondria-associated membrane (MAM) interface of endoplasmic reticulum (ER) and mitochondria.

Figure 1

Spectral features and in vitro contrast of ddGFP and ddYFP. (a) E. coli expressing ddGFP-A, ddGFP-B, or tdGFP-AB. (b) Absorbance and emission of ddGFP-A (black dashed line), ddGFP-B (gray line), and tdGFP-AB (solid black line). (c) Absorbance and (d) emission profiles of tdGFP-AB and its monomers before and after trypsinolysis. Inset shows the low intensity fluorescence signal. (e) E. coli expressing ddYFP-A, ddYFP-B, or tdYFP-AB. (f) Absorbance and emission profiles of ddYFP-A (gray line), ddYFP-B (black dashed line), and tdYFP-AB (solid black line). (g) Absorbance and (h) emission profiles of tdYFP-AB and its monomers before and after trypsinolysis. Inset shows the low fluorescence intensity signal.

Figure 2

Saturation binding curves for ddFP variants. (a) ddGFP-AB (solid green line) and ddGFP containing a K153E mutation in the ddGFP-B partner (dashed light green line). K_d values are 9 and 27 μM, respectively. (b) ddYFP-AB, K_d is 14.5 μM. Error bars are ± standard deviation for at least three independent experiments.

Figure 3

ddGFP labeling of MAM in HeLa cells. (a) MAM labeling strategy. (b) Expression of calnexin-ddGFP-A or (c) Tom20-ddGFP-B alone. (d) Coexpression of calnexin-ddGFP-A and Tom20-ddGFP-B. Cell nuclei (blue) are stained with DAPI. Scale bars, 10 μm.

Figure 4

ddGFP MAM label colocalizes with ER (right) and mitochondria (left). HeLa cells were transfected with three plasmids encoding calnexin-ddGFP-A, Tom20-ddGFP-B, and either a mCherry-ER or a mCherry-mitochondria marker. Top panel is ddGFP fluorescence, middle panel is red mCherry fluorescence, and bottom panel depicts merge. Images were acquired on an Olympus IX-81 motorized microscope spinning-disk confocal microscope. Scale bars, 10 μm.

Figure 5

The ddGFP probe is MAM-specific. (a) Wild-type (WT) or (b) mitofusin-2 knockout (Mfn-2 KO) MEFs cotransfected with calnexin-ddGFP-A and Tom20-ddGFP-B. Depicted are fluorescent images obtained for independent cotransfections. Cell nuclei (blue) are stained with DAPI. Scale bars, 10 μm.

Scheme 1. Conceptual Schematic of ddFP Technology^a

^aWeak or nonfluorescent protein monomers reversibly associate to form a fluorescent heterodimer. Chromophore indicated by starburst.