Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 8 (1), 5344

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process

Affiliations

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process

Tuğba Köker et al. Sci Rep.

Erratum in

Abstract

Many biotechniques use complementary split-fluorescent protein (sFPs) fragments to visualize protein-protein interactions, image cells by ensemble or single molecule fluorescence microscopy, or assemble nanomaterials and protein superstructures. Yet, the reassembly mechanisms of sFPs, including fragment binding rates, folding, chromophore maturation and overall photophysics remain poorly characterized. Here, we evolved asymmetric and self-complementing green, yellow and cyan sFPs together with their full-length equivalents (flFPs) and described their biochemical and photophysical properties in vitro and in cells. While re-assembled sFPs have spectral properties similar to flFPs, they display slightly reduced quantum yields and fluorescence lifetimes due to a less sturdy β-barrel structure. The complementation of recombinant sFPs expressed in vitro follows a conformational selection mechanism whereby the larger sFP fragments exist in a monomer-dimer equilibrium and only monomers are competent for fluorescence complementation. This bimolecular fragment interaction involves a slow and irreversible binding step, followed by chromophore maturation at a rate similar to that of flFPs. When expressed as fusion tags in cells, sFPs behave as monomers directly activated with synthetic complementary fragments. This study resulted in the development of sFP color variants having improved maturation kinetics, brightness, and photophysics for fluorescence microscopy imaging of cellular processes, including single molecule detection.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Spectral properties, fluorescence lifetime and brightness of split and full-length fluorescent proteins. (a) Absorption and emission spectra of split fluorescent proteins (sFPs). (b) Absorption and emission spectra of full-length fluorescent proteins (flFPs). (c) Comparison of fluorescence lifetime (τ) between split and full-length fluorescent proteins. ○: lifetime from the chromophore B-state, : lifetime from the chromophore A-state, *: lifetime from the chromophore I-state. (d) Comparison of brightness between all fluorescent proteins.
Figure 2
Figure 2
Photobleaching kinetic of complemented sGFP variants. (a) Model for irreversible photobleaching (FPible) and reversible photoconvertible dark state reactions (FPrble) for a native GFP (FPnat). (b) Normalized photobleaching kinetics for complemented sGFPori, sGFP1, sGFP2 and sGFP3. (c) Comparison of the forward (k1, left panel) and backward (k2, middle panel) photoconvertible dark state rate constants and of the irreversible photobleaching rate constants (k3, right panel) for all complemented sGFP variants.
Figure 3
Figure 3
Folding and maturation of full-length FPs. (a) Schematic models of FP folding and maturation processes. (b) Example of flGFP2 folding (red) and maturation (black) kinetics with tri-exponential and single exponential fits (green lines), respectively. (b) Comparison of kfold1, kfold2, kmat, and refolding efficiencies between all the flFP variants. SE: standard error of the fit. *: SE lower than three decimals are not reported.
Figure 4
Figure 4
Concentration-dependent dimer-monomer exchanges in recombinant split-fluorescent proteins. (a) Schematic of dimer-monomer equilibrium in recombinantly produced sFPs. Keq represents the equilibrium constant for dimer formation. (b) Size exclusion high-pressure liquid chromatography of non-complemented sGFP2 labeled with fluorescent ReAsH on an N-terminal tetracysteine tag. sGFP2 is mostly dimeric at 20 µM (apparent molecular weight of 61 KDa) but mostly monomeric at a lower concentration of 0.5 µM (apparent molecular weight of 31 KDa). The retention times of a set of calibrated molecular weight standards (68, 43, 29 and 14 KDa) are provided as reference. (c) Steady-state fluorescence anisotropy of ReAsH-labeled sGFP2 at different concentrations. The apparent equilibrium constant of dimer formation (Keq) is determined by fitting the anisotropy curve with the inset equation (green), which describes the ensemble anisotropy contributed by both dimer anisotropy (rd) and monomer anisotropy (rm) at each total sGFP2 concentration. Anisotropy values are presented as mean ± std from measurements in triplicate.
Figure 5
Figure 5
Assembly kinetics of split-fluorescent protein fragments. (a) Schematic of sFP complementation and maturation, including the sFP dimer-monomer equilibrium (Keq), an irreversible binding step of complementary M3 peptides to sFP monomers with rate constant kon and an irreversible chromophore maturation step with rate constant kmat. (b) Example of pseudo-first order fluorescence kinetic curves for increasing concentrations of sGFP2 incubated with 0.1 µM of complementary M3 peptides. Only one replicate out of three performed is shown for clarity. Under these conditions, the dimer-monomer equilibrium (orange dashes in (a)) does not affect the binding and maturation reactions (green dashes in (a)), allowing the observed binding rate kobs1 and maturation rate kobs2 to be determined at each sGFP2 concentration with a bi-exponential fit. (c) Distribution and fit (green) of kobs1 and kobs2 as a function of monomeric sGFP2 concentration to define the rate constants kon and kmat, respectively. kobs values are presented as mean ± std from measurements in triplicate. (d) Comparison of kon and kmat rate constants for some complemented sFPs. ND*: Not determined because sGFP3 exists as complexes bigger than dimers or monomers when expressed recombinantly. SE: standard error of the fit.
Figure 6
Figure 6
Live cell confocal imaging of complemented GPI-anchored split-FP fusions. (a) Schematic representation of GPI-sFP fusions expressed at the outer leaflet of the plasma membrane in U2OS cells. (b) Fluorescence confocal images of different complemented GPI-sFPs at the cell ventral plasma membrane (left) and corresponding differential interference contrast images. Scale bars: 10 μm.
Figure 7
Figure 7
Single molecule imaging and tracking of complemented GPI-anchored split-GFP protein fusions in cells. (a) Single frame TIRF image (left), full acquisition 2D-Gaussian super-resolved localizations (center) and reconstructed diffusion trajectories (right) for individual complemented GPI-sGFP2 at the plasma membrane of U2OS cells. Scale bar: 10 μm. (b) Comparison of individual molecule brightness for complemented GPI-sGFPori and GPI-sGFP2 at the plasma membrane. (c) Comparison of trajectory durations after single molecule tracking of GPI-sGFPori and GPI-sGFP2 at the cell plasma membrane.

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Kerppola TK. Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu. Rev. Biophys. 2008;37:465–487. - PMC - PubMed
    1. Cabantous S, et al. A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association. Sci. Rep. 2013;3:2854. doi: 10.1038/srep02854. - DOI - PMC - PubMed
    1. Cabantous S, Terwilliger TC, Waldo GS. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotechnol. 2005;23:102–107. - PubMed
    1. Huang Y-m, et al. Toward Computationally Designed Self-Reporting Biosensors Using Leave-One-Out Green Fluorescent Protein. Biochemistry. 2015;54:6263–6273. - PMC - PubMed
    1. Huang Y-m, Bystroff C. Complementation and Reconstitution of Fluorescence from Circularly Permuted and Truncated Green Fluorescent Protein. Biochemistry. 2009;48:929–940. - PMC - PubMed

Publication types

Feedback