A naturally monomeric infrared fluorescent protein for protein labeling in vivo

Abstract

Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.

Figure 1. Rational design of a naturally-monomeric infrared fluorescent protein

(a) Residues potentially involved in the dimer interface of DrBphP (PDB: 2O9B). (b) Sequence alignment of DrBphP and BrBphP. Residues in the core of dimer interface of DrBphP are colored in yellow, and the corresponding residues in BrBphP in cyan. (c) Excitation (blue) and emission (red) spectra of mIFP. (d) Analytical ultracentrifugation of mIFP at high concentrations (17 and 34 μM). Data were globally fitted to a function describing sedimentary equilibrium curves. Single-species fitting agrees well with the data over the entire range and yielded the molecular weight of mIFP 33.05 ± 0.98 kD, which is close to the expected molecular weight of a monomer (35.69 kD). (e) A structure model of mIFP with introduced mutations surrounding the chromophore BV (in purple). (f) Fluorescence of purified mIFP against pH. (g) Comparison of cellular brightness among IFP1.4, iRFP, and mIFP in live HeLa cells (infrared fluorescence normalized by co-expressed GFP under IRES). The standard deviation was calculated based on 20 cells for each IFP. (h) Representative fluorescence images of IFP1.4 (brightened 10 times), iRFP and mIFP in HeLa cells. 20 cells were examined for each IFP. Scale bar: 10 μm.

Figure 2. Expression of mIFP, histone or membrane fusions in model organisms

(a, b) Fluorescence image of Drosophila embryo expressing UAS-mIFP-histone 3.3 T2A HO1 driven by engrailed-GAL4. (b) High magnification view of the boxed area in a. (c) Fluorescence image of zebrafish eye expressing mIFP-H2B and HO1. (d-g) Two-color fluorescence imaging of Drosophila embryo expressing UAS-mIFP-histone 3.3 T2A HO1 and UAS-CD8-GFP, driven by elav-GAL4: (d) Whole embryo; (e-f) High magnification view of the boxed area in d. (e) GFP; (f) mIFP; (g) merged. (h-k) Three-color fluorescence imaging of Drosophila abdomen muscle expressing UAS-mIFP T2A HO1 driven by Mef2R-GAL4, Class IV DA neurons expressing CD4-tdTomato (ppk::CD4-tdTomato), extracellular matrix expressing GFP-viking (collagen): (h) tdTomato (in blue pseudo-color); (i) GFP; (j) mIFP; (k) merged. (l,m) Class IV DA neurons expressing CD4-mIFP in Drosophila larvae: (m) High magnification view of the boxed area in l. Scale bar, 50 μm (a); 10 μm (b); 20 μm (c); 50 μm (d); 10 μm (e-f); 20 μm (h-k); 50 μm (l); 20 μm (m).