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Split Fluorescent Protein Technology and (2) Present Five Case Studies How Chromotek’S Nano- Traps Can Be Applied to Tap the Full Potential of This Technology

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Split Fluorescent Protein Technology and (2) Present Five Case Studies How Chromotek’S Nano- Traps Can Be Applied to Tap the Full Potential of This Technology Michael Metterlein, Christian Linke-Winnebeck ChromoTek GmbH, Am Klopferspitz 19, 82152 Planegg-Martinried, Germany Abstract To understand the biology of proteins and with that cell function in health and disease, it is crucial to be able to visualize proteins in situ and to analyze their protein-protein interaction networks in vivo. To this end, fluorescent proteins (FPs) have proven an invaluable tool as protein tags and have literally thrown light upon many scientific questions. Fluorescent proteins were further developed into split FP variants, spurred by the need for dedicated tools for the analysis of protein-protein interactions. Here, (1) we give a review about split fluorescent protein technology and (2) present five case studies how ChromoTek’s Nano- Traps can be applied to tap the full potential of this technology. Summary 1. Jellyfish-derived GFP variants and most other fluorescent proteins from various species of coral or sea anemone share a common β-barrel fold composed of 11 single β-strands. Split fluorescent protein technology uses non-fluorescent fragments of this β-barrel, obtained by truncation between two or three β-strands to study single proteins or protein-protein interactions. Reconstitution of the full-length FP can be obtained either by conditioned (protein-protein interaction of fusion partners) or unconditioned (self- complementation) fragment association, recovering fluorescence. All aerobically grown cells and organisms that can be genetically modified have the potential to be used in split FP technology. 2. Split FP technology is frequently applied to cellular assays. ChromoTek’s Nano-Traps constitute attractive research tools for the biochemical validation of such experiments. For instance, the ChromoTek GFP-Trap has been successfully applied to different assay types such as protein self-complementation, BiFC, TriFC, or BiCAP, involving several different split GFP variants. Introduction A large number of fluorescent proteins Additionally, there are fluorescent proteins (FPs) has been discovered and developed that bind an exogenous chromophore (e.g. since the discovery of green fluorescent Halo, or Snap) but these are less frequently protein (GFP) in Aequorea victoria in 1962. used (Sanford and Palmer, 2017) and will Most FPs are derived from marine not be discussed here. For more details organisms such as jellyfish, coral, and sea about FPs in general, see also our anemone. These FPs bear an intrinsic informative ChromoBlog. chromophore. www.chromotek.com 1 Figure 1. Comparison of wtGFP with seven of its variants (eGFP, sfGFP, eYFP, Venus, BFP, CFP and eCFP) and two GFP homologs from other organisms (mCherry and mNeonGreen). A: Structural alignment of all selected FPs (except CFP), shown as cartoon presentation with the chromophore drawn as orange sticks in the center of the β-barrel. Color code and PDB IDs are listed in B. The structures were aligned using PDBeFold (http://www.ebi.ac.uk/msd-srv/ssm/) and adapted using PyMOL B: Overview of the selected FPs showing their mutations compared to wtGFP. C: Sequence identities in [%] between the selected FPs. “mNG” is mNeonGreen. The protein sequences were retrieved from http://www.fpbase.org, and sequence identities were calculated using the software Geneious. A vast number of fluorescent proteins has (confirmed by an overall root mean square been structurally characterized. Figure 1 deviation (rmsd) of only ~1.2 Å over 207 gives an overview of ten such fluorescent compared backbone Cα atoms). In proteins that have been selected because contrast, the shared sequence identity of their published use in split FP assays may be as low as 20-30 % (Figure 1C). (see above and Table 1). In practical terms, the sequence variety of These jellyfish-derived GFP variants (first fluorescent proteins derived from different eight in Figure 1B), mCherry from a sea organisms means that most anti-GFP anemone, and mNeonGreen from a antibodies exclusively bind to various sets lancelet share a highly conserved β-barrel of GFP variants derived from jellyfish. fold with a chromophore in its center. This Other FP homologs such as mCherry or chromophore is formed by three mNeonGreen require other, dedicated consecutive amino acids at positions 65, tools. ChromoTek offers such research 66, and 67 in an autocatalytical process tools for fluorescent proteins derived from called maturation. many different species. The high structural conservation between Protein-Protein Interaction Assays the ten selected FPs is illustrated by the Since the discovery of GFP, fluorescent structural alignment in Figure 1A proteins have become wildly successful 2 www.chromotek.com tools in biological and biomedical research, partners, the fragments of the reporter as recognized by the Nobel Prize in protein are brought into close proximity chemistry in 2008. The tagging of a protein and are thus able to reconstitute an active of interest (POI) by fusing it to a FP is widely protein. The reconstitution of a reporter used to visualize a POI’s subcellular protein from its fragments is also termed location in vivo or in vitro, to study its protein complementation. However, biochemistry (using methods such as protein complementation cannot only be immunoprecipitation), or to analyze mediated in a conditioned way by fluorescent cells by flow cytometry interacting fusion proteins (as just (Leonetti et al., 2016). described above), but also in an unconditioned way (self- Usually, it is not the single protein in complementation). The binding route isolation that is of interest, but rather its highly depends on the kind of split FP network of interactions with other fragments used. proteins. These protein-protein interactions (PPI) are fundamental to all The basic principle of protein processes of life, e.g. in signal transduction complementation can be traced back to or gene expression. Many methods have the 1950s, when Richards observed that been developed to study protein-protein subtilisin-cleaved fragments of interactions in cells, some of which are ribonuclease are active again after self- based on or can be used with (split) FPs. complementation (Kerppola, 2008). Since then, various protein complementation One of the most frequently applied assays using fragments of reporter methods is Förster resonance energy proteins such as β-galactosidase, TEV transer (FRET). It allows real-time detection protease, or luciferase have been of complex formation and dissociation. developed. However, the method has low sensitivity and works only if the fluorescent reporter However, these reporter proteins are not proteins are placed within 10 nm of each optimal for usage in protein-protein other (Kerppola, 2008, Miller et al., 2015). interaction assays, because the observed reporter enzyme products diffuse away Another PPI method is ChromoTek’s from the investigated protein-protein Fluorescent Two-Hybrid (F2H®) assay. This interaction site. This diffusion leads to assay enables real-time monitoring and impaired colocalization of marker activity quantitative analysis of interactions and PPI site (Kerppola, 2008, Cabantous et between GFP- and RFP-tagged proteins in al., 2013). By contrast, the use of live mammalian cells. fluorescent protein fragments as tags in Protein Complementation Assays protein complementation assays, first described by Ghosh and coworkers Protein-protein interactions can also be (Ghosh et al., 2000), enables studies with analyzed using protein complementation the marker activity inherent to the PPI site. assays. These assays all rely on the use of Ghosh and colleagues dissected a GFP fragments of a reporter protein, e.g. an variant (sg100GFP) between β-strands 7 enzyme or FP, that are fused to interacting and 8 and fused the resulting fragments to proteins. Upon interaction of the fusion strongly interacting antiparallel leucine www.chromotek.com 3 zippers. They revealed that reconstitution In 2003, Hu and Kerppola published of this split GFP variant is driven only by the designs for further variants of split FPs, interaction of both leucine zippers, works which were intended for the use in in vivo (e.g. E.coli) and in vitro (after GFP (multicolor) BiFC (Hu and Kerppola, 2003). fragment purification from inclusion They generated fragments of eGFP, eYFP, bodies), and that absorption and emission eCFP, and eBFP truncated either between maxima are identical to parental full-length β-strands 7 and 8 or between β-strands 8 GFP (Ghosh et al., 2000, Kerppola, 2008). and 9 (Figure 2). The resulting fragments and combinations thereof were tested as Progress in Split Fluorescent Protein fusions with interacting leucine zippers. Technology Several combinations of fragments yielded In the following, we will describe key indeed detectable fluorescence signals, developments in the field of split FP assays. but displayed varying tendencies for self- For an overview, commonly used split FPs complementation. Such self-binding is not are listed in Table 1; their split sites are desired in BiFC assays and can be mapped to a topology diagram of GFP in minimized by reducing expression of the Figure 2. fragment fusions to the lowest level that permits fluorescence detection. In the early 2000s, Hu and coworkers identified the first eYFP fragments that While split eYFP had been suggested for form active complements only upon usage in BiFC assays by Kerppola in 2008, binding of their interacting fusion partners. the eYFP variant Venus is recommended as They termed this mechanism of the split FP variant to use in
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