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Illustration of the split GFP scheme for the cell surface labeling of a GPCR protein.: (A) GFP structure, with GFP β-strands 1-9 (GFP1-9) colored in green and GFP β-strands 10-11 (GFP10-11) colored in gray. A trypsin cleavage site was introduced between the 9th and 10th β-strands, as indicated. (B) Schematic diagram for the preparation of GFP1-9. Following trypsin digestion, the two fragments were separated in the presence of 3 M guanidine hydrochloride. GFP1-9 was then purified and refolded. (C) Absorption and fluorescence emission spectra for wild type GFP, GFP1-9, and GFP10-11:GFP1-9 complex. (D) Schematic diagram for the assembly between GFP1-9 and GFP10-11 engineered to the third extracellular loop of GPR17, a GPCR protein.
Source publication
Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrat...
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Citations
... Premature chromophore formation can also take place prior to self-assembly when the superfolder GFP is split into GFP1-9 and GFP10-11 fragments. Through the introduction of a protease cleavage site between the 10th and 11th β-strands or the 9th and 10th β-strands, GFP1-10 [31] or GFP1-9 [32] with a pre-matured chromophore can be obtained by cleaving the superfolder GFP using a protease. Similarly, the prematured GFP1-10 or GFP1-9 can rapidly complement GFP11 or GFP10-11, leading to the generation of a fluorescence signal [32]. ...
... Through the introduction of a protease cleavage site between the 10th and 11th β-strands or the 9th and 10th β-strands, GFP1-10 [31] or GFP1-9 [32] with a pre-matured chromophore can be obtained by cleaving the superfolder GFP using a protease. Similarly, the prematured GFP1-10 or GFP1-9 can rapidly complement GFP11 or GFP10-11, leading to the generation of a fluorescence signal [32]. These self-assembled GFP fragments have been widely applied in various fields, including the analysis of protein topology and subcellular localization [33], investigations into protein solubility [34]. ...
... These self-assembled GFP fragments have been widely applied in various fields, including the analysis of protein topology and subcellular localization [33], investigations into protein solubility [34]. Jiang et al. [32] employed the split-GFP (GFP1-9/GFP10-11) for qualitative imaging of cell surface labelling of G proteincoupled receptors, but the number of displayed proteins was not quantified. ...
Background
Microbial cell surface display technology allows immobilizing proteins on the cell surface by fusing them to anchoring motifs, thereby endowing the cells with diverse functionalities. However, the assessment of successful protein display and the quantification of displayed proteins remain challenging. The green fluorescent protein (GFP) can be split into two non-fluorescent fragments, while they spontaneously assemble and emit fluorescence when brought together through complementation. Based on split-GFP assembly, we aim to: (1) confirm the success display of passenger proteins, (2) quantify the number of passenger proteins displayed on individual cells.
Results
In this study, we propose two innovative methods based on split-green fluorescent protein (split-GFP), named GFP1-10/GFP11 and GFP1-9/GFP10-11 assembly, for the purpose of confirming successful display and quantifying the number of proteins displayed on individual cells. We evaluated the display efficiency of SUMO and ubiquitin using different anchor proteins to demonstrate the feasibility of the two split-GFP assembly systems. To measure the display efficiency of functional proteins, laccase expression was measured using the split-GFP assembly system by co-displaying GFP11 or GFP10-11 tags, respectively.
Conclusions
Our study provides two split-GFP based methods that enable qualitative and quantitative analyses of individual cell display efficiency with a simple workflow, thus facilitating further comprehensive investigations into microbial cell surface display technology. Both split-GFP assembly systems offer a one-step procedure with minimal cost, simplifying the fluorescence analysis of surface-displaying cells.
... In addition, mutational analyses of key residues involved in either BNIP3L/NIX dimerization or in BNIP3L/NIX phosphorylation were demonstrated to affect mitochondrial clearance upon CCCP treatment [185]. Consequently, this dimerization event could be exploited as a mitophagy readout, potentially through a split-green fluorescent protein (GFP) system to monitor receptor homodimer formation in single cells [186,187]. This approach has been used to successfully tag members of the G-protein coupled receptor (GPCR) family of cell surface receptors [188]. ...
The best-known hallmarks of Parkinson’s disease (PD) are the motor deficits that result from the degeneration of dopaminergic neurons in the substantia nigra. Dopaminergic neurons are thought to be particularly susceptible to mitochondrial dysfunction. As such, for their survival, they rely on the elaborate quality control mechanisms that have evolved in mammalian cells to monitor mitochondrial function and eliminate dysfunctional mitochondria. Mitophagy is a specialized type of autophagy that mediates the selective removal of damaged mitochondria from cells, with the net effect of dampening the toxicity arising from these dysfunctional organelles. Despite an increasing understanding of the molecular mechanisms that regulate the removal of damaged mitochondria, the detailed molecular link to PD pathophysiology is still not entirely clear. Herein, we review the fundamental molecular pathways involved in PINK1/Parkin-mediated and receptor-mediated mitophagy, the evidence for the dysfunction of these pathways in PD, and recently-developed state-of-the art assays for measuring mitophagy in vitro and in vivo.
... Both dimers purified here were tethered via cytosolic C termini. Although we have not tested N-terminal or extracellular tethers, we would expect these to work based on successful complementation of sGFP in other topological contexts (e.g., (25)(26)(27)(28)). Other sGFP constructs may yield additional flexibility in construct design: sGFP10-11 inserted into a loop internal to a fusion protein sequence will complement separately expressed GFP strands 1 to 9 (25,28). ...
... Although we have not tested N-terminal or extracellular tethers, we would expect these to work based on successful complementation of sGFP in other topological contexts (e.g., (25)(26)(27)(28)). Other sGFP constructs may yield additional flexibility in construct design: sGFP10-11 inserted into a loop internal to a fusion protein sequence will complement separately expressed GFP strands 1 to 9 (25,28). This suggests that sGFP10-11 does not need to be at a terminus of a tethered receptor, providing a way to avoid the truncation of a terminus (e.g., that of LRP6) while minimizing linker length for efficient parallel reconstitution. ...
... For Fzd4 quantification, the standard curve consisted of Fzd4 in detergent at 0.5, 1, 2, 4, and 8 nM, with 10 μl loaded per lane. All His-tag standards were combined on the same gel, with lanes containing 10 μl each of 12.5, 25, 50, 100, and 200 nM LRP6, as well as 25,50,100,200, and 400 nM each MSP. Nanodisc samples were typically diluted fourfold for the anti-His blot and 100-fold for the anti-FLAG blot to fall within the linear range of the standard curves. ...
Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here we describe a technique to obtain purified, homogeneous membrane protein dimers in nanodiscs using a split GFP tether. Complementary split GFP tags associate to tether the co-expressed dimers and control both stoichiometry and orientation within the nanodiscs, as assessed by quantitative western blotting and negative stain electron microscopy. The split GFP tether confers several advantages over other methods: it is highly stable in solution and in SDS-PAGE, which facilitates screening of dimer expression and purification by fluorescence, and also provides a dimer-specific purification handle for use with GFP nanobody-conjugated resin. We used this method to purify a Frizzled-4 (Fzd4) homodimer and a Fzd4/low-density lipoprotein receptor-related protein 6 (LRP6) heterodimer in nanodiscs. These examples demonstrate the utility and flexibility of this method, which enables subsequent mechanistic molecular and structural studies of membrane protein pairs.
... Here, we report the dimeric structural model of GPR17 in the cell membrane. Using a split-GFP labeling strategy we have developed [38], we obtained multiple FRET distances for the modeling of the GPR17 dimer structure. The structural model is well converged and can be validated by mutations at the interface. ...
... We fused a GFP or mCherry protein at the N terminus of GPR17, as fluorescence donor and acceptor, respectively (Figure 2(a)). Additionally, using a split-GFP surface labeling strategy [38], we introduced GFP at one of the extracellular loops of a GPR17 protein as a fluorescence donor (Figure 2(a)). In detail, β10 and β11 stands derived from split-GFP were inserted to ECL1, ECL2, or ECL3. ...
... A "cap" comprising β1-β9 from the split-GFP is subsequently added to label the GPR17 expressing at the cell membrane. The surface labeling may have an impact on the ligand-binding property of GPR17 [38]. Nevertheless, we focused on the quaternary interaction of GPR17 protomers in the unliganded state. ...
Class-A G protein-coupled receptors (GPCRs) are known to homo-dimerize in the membrane. Yet, methods to characterize the structure of GPCR dimer in the native environment are lacking. Accordingly, the molecular basis and functional relevance of the class-A GPCR dimerization remain unclear. Here, we present the dimeric structural model of GPR17 in the cell membrane. The dimer mainly involves transmembrane helix 5 (TM5) at the interface, with F229 in TM5, a critical residue. An F229A mutation makes GPR17 monomeric regardless of the expression level of the receptor. Monomeric mutants of GPR17 display impaired ERK1/2 activation, and cannot be properly internalized upon agonist treatment. Conversely, the F229C mutant is cross-linked as a dimer and behaves like wild-type. Importantly, the GPR17 dimer structure has been modeled using sparse inter-protomer FRET distance restraints obtained from fluorescence lifetime imaging microscopy. The same approach can be applied to characterizing the interactions of other important membrane proteins in the cell.
... The antibody single chain variable region fragments (scFvs) fused with the evolved GFP11 at the C-terminus showed great solubility and folding efficiency, which were widely employed as fluorescencetagged recognition modules in different assays, such as fluorescence linked immunosorbant assay, flow cytometry, and yeast display [36]. Another application is specific labeling of G protein coupled receptors (GPCRs) on cell surface for monitoring their internalization processes [37]. GFP10-11 was inserted into the third loop of a GRCR protein, GPR17. ...
Fluorescent proteins (FPs) are powerful and fundamental tools in bio-analytical research, ranging from tracking biological molecules to monitoring dynamic processes in living cells. Currently, great efforts have been made on structurally engineering of FPs with new fluorescent signal-switch mechanisms via rational or semi-rational design. In this review, we summarize a variety of strategies for redesigning FPs, including circularly permuted FPs, split FPs, supercharged FPs, and unnatural amino acid-containing FPs, as well as RNA/DNA mimics of FPs. We focus on the design strategies and target-responsive mechanisms of the engineered FPs and their current applications in biosensing and bioimaging. The recent advances summarized here reveal that the great flexibility of the structure and the outstanding designability endow FPs with remarkable potential as versatile sensing elements for biochemical analysis.
... Following these observations, several studies have exploited the prematuration of the chromophore in order to improve split-GFP kinetics. These include the production of prematured version of GFP1-10 or GFP1-9 by incorporating a proteolytic site in the GFP scaffold that release strand β11 [113,114] or β10-11 [116]. Alternative approaches involve coexpressing both fragments and inducing their disassembly under denaturation conditions [114], or purifying each fragment separately under acid-pH conditions [115]. ...
Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.
... As such, the FLIM-FRET provides a 121 more accurate distance measurement between fluorophores than the standard intensity-based 122 FRET measurement. In addition to N-terminal labeling, we also introduced fluorophores at the 123 extracellular loops (ECLs) of GPR17 using a split-GFP strategy (Jiang et al., 2016), and 124 measured multiple FRET distances. Using the distance restraints, we have obtained a well-125 converged structural model of GPR17 homo-dimer in the cell membrane and identified 126 TM5/TM6 as the dimer interface. ...
G protein coupled receptors (GPCRs) have been shown homo-dimeric. Despite extensive studies, no single residue has been found essential for dimerization. Lacking an efficient method to shift the monomer-dimer equilibrium also makes functional relevance of GPCR dimer elusive. Here, using fluorescence lifetime-based imaging for distance measurements, we characterize the dimeric structure of GPR17, a class A GPCR, in cells. The structure reveals transmembrane helices 5 and 6 the dimer interface, and pinpoints F229 a key residue, mutations of which can render GPR17 monomeric or dimeric. Using the resulting mutants, we show that GPR17 dimer is coupled to both Gα i and Gα q signaling and is internalized, whereas GPR17 monomer is coupled to Gα i signaling only and is not internalized. We further show that residues equivalent to F229 of GPR17 in several other class A GPCRs are also important for dimerization. Our findings thus provide fresh insights into GPCR structure and function.
... With protein-based therapeutics in mind, Bale et al. (6) targeted nanoparticles to specific organelles in live cells. As imaging tools, these split FP pairs help visualize endogenous localization patterns and dynamics of proteins in live cells both with standard and super-resolution microscopy (33,49,51,53,77,105). In another application from the Pinaud lab, split GFP links together two gold nanoparticles, with a mature GFP complex acting as the bridge, creating a photoacoustic and surface-enhanced Raman spectroscopy imaging probe. ...
Many proteins can be split into fragments that spontaneously reassemble, without covalent linkage, into a functional protein. For split green fluorescent proteins (GFPs), fragment reassembly leads to a fluorescent readout, which has been widely used to investigate protein–protein interactions. We review the scope and limitations of this approach as well as other diverse applications of split GFPs as versatile sensors, molecular glues, optogenetic tools, and platforms for photophysical studies. Expected final online publication date for the Annual Review of Biophysics Volume 48 is May 3, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Complementation of split GFP reporters (and its variants) was already used to visualize protein-protein interaction in various eukaryotic systems including mammalian cells. Association of several split GFP pairs was known to fluoresce [16][17][18][19] but their fluorescence dictated by the The Nterminal luminal domain has a Ca 2+ binding EF hand and a sterile alpha motif (SAM). The C-terminal cytoplasmic fragment has two coiled-coils (CC1 and self-assembly and maturation of split pairs apparently vary with the split architecture [20]. ...
... Cells adhered to coverslips were treated with TG for 30mins before imaging. This duration is sufficient for the self-assembly of complementary eGFP split pairs to form a fully matured fluorescent eGFP molecule [19]. ...
Several signaling proteins require self-association of individual monomer units to be activated for triggering downstream signaling cascades in cells. Methods that allow visualizing their underlying molecular mechanisms will immensely benefit cell biology. Using enhanced Green Fluorescent Protein (eGFP) complementation, here I present a functional imaging approach for visualizing the protein-protein interaction in cells. Activation mechanism of an ER (endoplasmic reticulum) resident Ca2+ sensor, STIM1 (Stromal Interaction Molecule 1) that regulates store-operated Ca2+ entry in cells is considered as a model system. Co-expression of engineered full-length human STIM1 (ehSTIM1) with N-terminal complementary split eGFP pairs in mammalian cells fluoresces to form 'puncta' upon a drop in ER lumen Ca2+ concentration. Quantization of discrete fluorescent intensities of ehSTIM1 molecules at a diffraction-limited resolution revealed a diverse set of intensity levels not exceeding six-fold. Detailed screening of the ehSTIM1 molecular entities characterized by one to six fluorescent emitters across various in-plane sections shows a greater probability of occurrence for entities with six emitters in the vicinity of the plasma membrane (PM) than at the interior sections. However, the number density of entities with six emitters was lesser than that of others localized close to the PM. This finding led to hypothesize that activated ehSTIM1 dimers perhaps oligomerize in bundles ranging from 1-6 with an increased propensity for the occurrence of hexamers of ehSTIM1 dimer units close to PM even when its partner protein, ORAI1 (PM resident Ca2+ channel) is not sufficiently over-expressed in cells. The experimental data presented here provide direct evidence for luminal domain association of ehSTIM1 monomer units to trigger activation and allow enumerating various oligomers of ehSTIM1 in cells.
