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DOE OSTI · 3007513

Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions

Groseclose, Thomas Michael [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000178314294)·Kober, Erin [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)]·Zupancic, Jennifer Marie [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000295225338)·McLelland, Claire K. [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)]·Esmith Lujan, Lexy Amber [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)]·Kunde, Yuliya A. [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:000000033252141X)·Mozden, Sarah C. [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)]·Velappan, Nileena [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000244889126)·Lillo, Antonietta Maria [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000204576598)·Solomon, Emilia [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)]·Yoder, Jacob [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)]·Waldo, Geoffrey S. [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)]·Nguyen, Hau Thi Bich [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000301749622)
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Abstract

Protein-protein interactions (PPIs) are critical to a range of biological processes and, consequently, aberrant interactions are implicated in many disorders. The study of the complex networks of PPIs promises to elucidate undiscovered roles in cellular processes and the mechanisms of disease. To accomplish this, tools to effectively sense PPIs are necessary. Effective PPI sensors must rapidly detect interactions in real-time with high sensitivity without perturbing the proteins of interest (POIs) under study. Split fluorescent proteins have previously been used to successfully monitor PPIs, in part due to the small size of the tags. Here, we developed an optimized tripartite split GFP system based on Corynactis californica GFP (ccGFP) to detect PPIs in vitro. In this sensor system, ccGFP fragments ccGFP10 and ccGFP11 are tagged to two POIs. PPIs can then be detected via fluorescence by complementation to the third fragment, ccGFP1-9, which reconstitutes functional ccGFP. The optimized ccGFP system shows improved detection kinetics and pH and temperature stability compared to a previous system. We then validated the sensor by monitoring PPIs in two model systems: attractive/repulsive coiled-coils and rapamycin-inducible FRB/FKBP heterodimerization. Finally, we developed an anti-tripartite ccGFP single-chain variable fragment (scFv), which could enable versatile detection of identified protein-protein complexes.

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59 BASIC BIOLOGICAL SCIENCESAntibodyDirected evolutionGreen fluorescent protein (GFP)Protein detectionProtein engineeringProtein fragment complementationProtein taggingProtein-protein interactionsSingle-chain variable fragment (scFv)Split fluorescent protein

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Groseclose, Thomas Michael [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000178314294), Kober, Erin [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)], Zupancic, Jennifer Marie [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000295225338), McLelland, Claire K. [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)], Esmith Lujan, Lexy Amber [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)], Kunde, Yuliya A. [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:000000033252141X), Mozden, Sarah C. [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)], Velappan, Nileena [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000244889126), Lillo, Antonietta Maria [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000204576598), Solomon, Emilia [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)], Yoder, Jacob [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)], Waldo, Geoffrey S. [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)], Nguyen, Hau Thi Bich [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)] (ORCID:0000000301749622). 2025-11-19. Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions. https://doi.org/10.1038/s41598-025-24657-6

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