Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization

Abstract

When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true- from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased sensitivity and renders analysis of weak or transient interactions difficult to perform. In this work, we describe the development of reporters that can be chemically induced to dimerize independently of the investigated interactions and thus alleviate these issues. We incorporated our reporters into the widely used split ubiquitin-, bimolecular fluorescence complementation (BiFC)- and Förster resonance energy transfer (FRET)- based methods and investigated different protein-protein interactions in yeast and plants. We demonstrate the functionality of this concept by the analysis of weakly interacting proteins from specialized metabolism in the model plant Arabidopsis thaliana. Our results illustrate that chemically induced dimerization can function as a built-in control for split-based systems that is easily implemented and allows for direct evaluation of functionality.

Specificity of MYB interactions relies on motifs in ordered and disordered contexts

“…Full-length MYB29 and MYB75 encoding altered motifs were ordered as synthetic genes from General Biosystems (NC, USA), shorter mutated MYB29 versions for MIM characterisation were ordered as gene fragments from Twist Bioscience (CA, USA), full-length MYB29 encoding proteins with the amino acid residue substitutions L190A or L190V were ordered as synthetic genes from Twist Bioscience, and all were then subcloned into the respective vectors. Constructs for split-ubiquitin assays were generated by amplifying template DNA with primers to add SfiI overhangs compatible with directional cloning into the pFRB (prey) and pFKBP12 (bait) vectors (35). For the expression of MYB29-WT, MYB29-L190A and MYB29-L190V in A. thaliana , the pFRU35S plasmid was generated from a derivative of the P2P3 double Gateway vector (Invitrogen), where the antibiotic selection sequence was replaced by the cassette for selection of transformants by fluorescence microscopy of the pFAST-R05 plasmid (36).…”

Unravelling Protein-Protein Interaction Networks Linked to Aliphatic and Indole Glucosinolate Biosynthetic Pathways in Arabidopsis

“…A plant expression cassette containing a ubiquitin (UBQ10) promoter and a RBC terminator were PCR amplified and inserted into the pEAQ-HT (Sainsbury et al, 2009 ) vector backbone. The coding sequences of mTurquoise2 (Goedhart et al, 2012 ) or mVenus were inserted into the resulting vector (Andersen et al, 2016 ). The respective coding sequences were PCR amplified with primer pairs CYP83A1_FW/CYP83A1_NSR, CYP83B1_FW/CYP83B1_NSR, At1G04340_FW/At1G04340_NSR, At4G14420_FW/At4G14420_NSR, and At5G43460_FW/At5G43460_NSR (Table S1 ) and inserted into the USER™ cassette of the fluorescent protein tagging vectors.…”

“…In addition, UGT74B1 enzyme kinetics support a channeling mechanism to achieve efficient reaction speed at physiological substrate concentration and to avoid product inhibition (Kopycki et al, 2013 ). Indeed, investigations of specific protein-protein interactions within the pathway have revealed interactions between the UGT74 and SOT enzymes, independently confirmed by yeast 2-hybrid, bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET) measurements (Andersen et al, 2016 ).…”

Developing High Sensitivity/Specificity Detection Systems for Studying Protein Interactions

“…One such method termed DNA-PAINT can achieve spatial DNA-PAINT achieves both high specificity and high number of usable fluorophores the technique involves stochastic switching of on and off states using fluorescence. The on/off switch is achieved via fluorescently labelled oligonucleotides called imager strands that bind to complementary docking strands of DNA nanostructures through repetitive and transient binding [71][72][73][74][75][76][77][78][79][80][81][82][83][84][85]. Spatial resolution is achieved within 25 nm and the technique has been used for multicolour sub diffraction.…”