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.

A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far‐Red Fluorescent BDFP1.6

“…The above design of the linkers may provide additional benefits for subsequent quantitative FRET analysis, which confines the conformational changes within fusion proteins, and thus, resulting in a stable FRET signal. These results show the great potential of linker engineering for improving the fluorescence intensity and related effective brightness (Figure , below) of fluorescent fusion proteins, in addition to engineering FPs themselves, in various biological and biomedical research studies and applications …”

“…In most situation, linkers can improve activities and achieve the structural flexibility of fusion proteins; most current linkers have loop structures . First, BDFP1.2 was fused to the N terminus of mCherry by linker1 or linker2 (Table S1), and the fluorescence emission at λ =666 nm had a slight increase (Figure S1).…”

“…As a result, the FRET signal of the linker2‐ligated fusion is better, possibly due to the increase in the distance resulting from the introduction of a helix (Table S1). Compared with linker3, the efficiency is still low. Then we designed linker4, which consisted of two β‐sheets and one random coil (Figure and Table S1).…”

Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways

“…To get non-fluorescent tagged proteins, the open reading frames of NEDD8 and NEDD8 were extracted by SalI/NotI digestion and cloned into the SalI/NotI sites of the pET28(b) vector. CyPet-linker3-NEDD8 was cloned as described 18 . We used full-length cDNA for all NEDD8, APPBP1, UBA3 and Ubc12.…”

“…We previously developed novel highly sensitive and quantitative FRET tools to determine the affinities of protein-protein interactions and the dynamics and specificities of enzymatic reactions in the SUMO 16 , the ubiquitin 17 , and the NEDD8 18 enzymatic cascades. Here we report d, for the first time, an unanticipated discovery that, one subunit of NEDD8 E1 activating enzyme, APPBP1, can be dispensable in NEDD8 activation, in contrast to that of SUMOylation, which both subunits of E1 activating enzyme, Aos1 and Uba2 are absolutely required for SUMO activation by using the quantitative FRET technology.…”