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

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Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology International Journal of Molecular Sciences Review Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology Jean-Denis Pedelacq 1,* and Stéphanie Cabantous 2,* 1 Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France 2 Centre de Recherche en Cancérologie de Toulouse (CRCT), Inserm, Université Paul Sabatier-Toulouse III, CNRS, 31037 Toulouse, France * Correspondence: [email protected] (J.-D.P.); [email protected] (S.C.) Received: 15 June 2019; Accepted: 8 July 2019; Published: 15 July 2019 Abstract: 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. Keywords: fluorescent protein; superfolder; split-GFP; bipartite; tripartite; folding; PPI 1. Superfolder Fluorescent Proteins: Progenitor of Split Fluorescent Protein (FP) Systems Previously described mutations that improve the physical properties and expression of green fluorescent protein (GFP) color variants in the host organism have already been the subject of several reviews [1–4] and will not be described here. A centralized database with hundreds of fluorescent proteins (FPs) was recently developed (https://www.fpbase.org/), which provides researchers with easy access to up-to-date information [5]. In this chapter, we describe the most recent advances in superfolder protein engineering and their application in living cells and animals. 1.1. Improving the Folding of avGFP The Aequorea victoria GFP or avGFP was cloned in 1992 by Prasher and colleagues [6]. It comprises 238 amino acids that adopt an 11-stranded barrel structure, known as “β-can”, with a central helix containing the chromophore [7]. The chromophore consists of three amino acid residues (Ser 65, Tyr 66, and Gly 67) that undergo cyclisation, dehydration, and oxidation during maturation. The maturation of the chromophore and therefore the emission of fluorescence requires proper GFP folding [7]. The auto-catalytic post-translational modifications of Ser 65, Tyr 66, and Gly 67 can occur when the protein is expressed in prokaryotic or eukaryotic species [8]. avGFP has a maximum excitation peak at 396–398 nm (corresponding to the neutral state of Tyr 66 in the chromophore) and a lower secondary peak at 476–478 nm (corresponding to the deprotonated anionic state of Tyr 66) [9,10], the emission peak is at 510 nm [9]. Thus, the excitation of avGFP by the 488 nm line of the Argon laser (standard in microscopy and flow cytometry) indicates low fluorescence intensity levels. avGFP was Int. J. Mol. Sci. 2019, 20, 3479; doi:10.3390/ijms20143479 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, x 2 of 19 Int. J. Mol. Sci. 2019, 20, 3479 2 of 19 avGFP was then evolved by site-directed mutagenesis to optimize brightness and excitation efficiencythen evolved at 488 by nm. site-directed The S65T mutagenesismutation accelera to optimizetes chromophore brightness formation and excitation and promotes efficiency hydrogen at 488 nm. bondsThe S65T that mutation stabilize the accelerates anionic state, chromophore resulting formation in a unique and excitation promotes peak hydrogen at 489 nm bonds and thata brightness stabilize 5the times anionic higher state, than resulting that of in aavGFP unique [11]. excitation In addition, peak at the 489 F64L nm and mutation a brightness improves 5 times chromophore higher than maturationthat of avGFP at 37 [11 °C]. In[12]. addition, Subsequently, the F64L the mutation gene sequence improves of chromophorethe corresponding maturation S65T/F64L at 37 double◦C[12]. mutantSubsequently, was converted the gene to sequencehuman codons of the to correspondingform the “enhanced-GFP” S65T/F64L double(eGFP), mutantwhich combines was converted a high expressionto human codonsrate in mammalian to form the cells “enhanced-GFP” and a fluorescen (eGFP),ce intensity which combines30 times higher a high than expression that of avGFP rate in [12].mammalian cells and a fluorescence intensity 30 times higher than that of avGFP [12]. RecombinantRecombinant avGFP avGFP produced produced in in EscherichiaEscherichia coli coli isis poorly poorly soluble soluble and and even even improved improved GFP GFP variantsvariants like like the the folding folding reporter reporter GFP GFP or or frGFP frGFP [13], [13], which which includes includes folding folding mutations mutations F99S, F99S, M153T, M153T, andand V163A V163A [14] [14] in in addition addition to to F64L F64L and and S65T S65T [12], [12], often often exhibit exhibit folding folding defects defects when when fused fused to to other other proteins.proteins. A A molecular molecular evolution evolution strategy strategy was was appli applieded to to frGFP frGFP to to create create a a superfolder superfolder GFP GFP (sfGFP) (sfGFP) variantvariant whose whose folding folding is is not affected affected by fusion to poorly foldedfolded proteinsproteins [[15].15]. This This new new variant variant incorporatesincorporates sixsix mutationsmutations compared compared to to frGFP frGFP (Figure (Figure1) and 1) isand more is resistantmore resistant to chemical to chemical and thermal and thermaldenaturation, denaturation, with folding with kineticsfolding fivekinetics times five faster times than faster that than of frGFP that [of15 ]frGFP and ten [15] times and fasterten times than fasterthat of than the “Venus”that of the GFP “Venus” variant GFP [16]. variant The high-resolution [16]. The high-resolution structures of structures frGFP and of sfGFPfrGFP highlightedand sfGFP highlighteda major role ofa themajor S30R role mutation of the onS30R the stabilizationmutation on of the the βstabilization-barrel through of the formationβ-barrel through of a network the formationof electrostatic of a network interactions of electrostatic [15]. Additional interactions mutations [15]. have Additional been identified mutations that have replace been hydrophobic identified thatresidues replace at hydrophobic the dimer interface residues with at the positively dimer interf chargedace with residues positively to force charged the residues monomerization to force the of monomerizationsfGFP [17] and other of sfGFP FPs for[17] optimal and other performance FPs for optimal as fusion performance tags [18]. as fusion tags [18]. Figure 1. Superfolder variants of (top) green fluorescent protein (GFP) and (bottom) DsRed. The β-can Figure 1. Superfolder variants of (top) green fluorescent protein (GFP) and (bottom) DsRed. The β-can structuresstructures of ofGFP GFP (PDB (PDB code code 1ema) 1ema) and and DsRed DsRed (PDB (PDB code code 1g7k) 1g7k) share share ~23% sequence identity.identity. StrandsStrands β1β to1 toβ11β11 are are colored colored from from dark dark red red to to magenta magenta in in th thee ribbon structures and correspondingcorresponding tables.tables. MutationsMutations refer refer to to A.A. victoria victoria GFPGFP (avGFP) (avGFP) and and DsRed DsRed indicated indicated on on a a black background. The asterisk (*)(*) • refersrefers to tothe the ratiometric ratiometric pHluorin. pHluorin. ( () The) The Q80R Q80R mutation mutation is is present present in in most most cDNA constructs andand maymay havehave resulted resulted from from a PCR a PCR error error [19]. [19 ]. 1.2. Superfolder Color Variants and Applications 1.2. Superfolder Color Variants and Applications Experiments carried out on the extreme thermophile Thermus thermophilus (Tth), have shown that Experiments carried out on the extreme thermophile Thermus thermophilus (Tth), have shown sfGFP, and not eGFP, could serve as a tool in cell localization experiments [20]. Given the exceptional that sfGFP, and not eGFP, could serve as a tool in cell localization experiments [20]. Given the stability of sfGFP, its tolerance to circular permutations was examined by cutting out loops connecting β exceptional stability of sfGFP, its tolerance to circular permutations was examined by cutting out strands while rewiring the original N- and C-terminal extremities. Circular permutants (cp) from sfGFP loops connecting β strands while rewiring the original N- and C-terminal extremities. Circular are mostly soluble with fluorescence yields higher than those measured from frGFP [15]. The most permutants (cp) from sfGFP are mostly soluble with fluorescence yields higher than those measured favorable cp provided a basis for testing protein insertion and creating genetically encoded sensors from frGFP [15]. The most favorable cp provided a basis for testing protein insertion and creating to study glutamate events in neurobiology [21], for cytosolic and cell surface ATP imaging [22], genetically encoded sensors to study glutamate events in neurobiology [21], for cytosolic and cell and neuronal
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