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

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.

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

“…Alternatively, for systems intentionally evolved to self-assemble, such as GFP1–10 and GFP11, the arguably more relevant parameter to assess fragment complementation is the on-rate (note that the equilibrium constant equals the ratio between the on-rate and off-rate). For fragment pairs that contain intermediates en route to a mature chromophore, on-rates for different FP variants range from 25 to 70 M −1 s −1 (70). For fragments already containing a mature chromophore, the few reported on-rates are one to two orders of magnitude larger (25, 26, 46, 58).…”

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

“…Still, a better understanding of the folding of GFP1–10 in vitro could help improving this self-assembly process. GFP1–10 was shown to partition between monomeric and dimeric forms [39,111]. Further in-depth analysis of the assembly of these split-GFP fragments indicated that the oligomerization state of GFP1–10 is important for association with GFP11, suggesting a more favorable binding of GFP11 to monomeric GFP1–10 [111].…”

“…GFP1–10 was shown to partition between monomeric and dimeric forms [39,111]. Further in-depth analysis of the assembly of these split-GFP fragments indicated that the oligomerization state of GFP1–10 is important for association with GFP11, suggesting a more favorable binding of GFP11 to monomeric GFP1–10 [111]. Therefore, we lack information on how the design of a monomeric GFP1–10 variant could improve the kinetic properties of the split-GFP association.…”

Live-cell imaging of single mRNA dynamics using split superfolder green fluorescent proteins with minimal background

“…Therefore, we cannot simply attribute the better performance of the tripartite sfGFP system to the difference between binary and ternary interactions. It is possible that other improved bipartite split FP systems (Huang et al 2015;Feng et al 2017;Köker et al 2018) may perform as well as the tripartite sfGFP system tested in this report.…”