title: A linker strategy for trans-FRET assay to determine activation intermediate of NEDDylation cascade.
abstract: Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a valuable tool for elucidating molecular interactions in vitro and in vivo. Quantitative FRET analysis is a powerful method for determining biochemical parameters and molecular distances at nanometer levels. Recently, we reported theoretical developments and experimental procedures for determining the dissociation constant, Kd and enzymatic kinetics parameters, Kcat and KM, of protein interactions with the engineered FRET pair, CyPet and YPet. The strong FRET signal from this pair made these developments possible. However, the direct link of fluorescent proteins with proteins of interests may interfere with the folding of some fusion proteins. Here, we report a new protein engineering strategy for improving FRET signals by adding a linker between the fluorescent protein and the targeted protein. This improvement allowed us to follow the covalent conjugation of NEDD8 to its E2 ligase in the presence of E1 and ATP, which was difficult to determine without linker. Three linkers, LAEAAAKEAA, TSGSPGLQEFGT, and LAAALAAA, which are alpha helix or random coil, all significantly improved the FRET signals. Our results show a general methodology for improving trans-FRET signals to effectively determine biochemical reaction intermediates.
authors.affiliation: Department of Bioengineering, Center for Bioengineering Research, Bourns College of Engineering, 900 University Avenue, Riverside, California, 92521.
authors.forename: Harbani Kaur
authors.initials: HK
authors.lastname: Malik-Chaudhry
authors.forename: Amanda
authors.initials: A
authors.lastname: Saavedra
authors.forename: Jiayu
authors.initials: J
authors.lastname: Liao
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D000075687:NEDD8 Protein; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D000075687:NEDD8 Protein; C513172:Nedd8 protein, mouse; C583290:Nedd8-activating enzyme E1 protein, mouse; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes
keywords: Förster resonance energy transfer (FRET); enzymatic specificity; peptide linker; reaction intermediates; ubiquitination, sumoylation