Impact of fluorescent protein fusions on the bacterial flagellar motor

Abstract

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation— new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.

Catch bond drives stator mechanosensitivity in the bacterial flagellar motor

“…A previous measurement of ( 10 ), performed on immobilized cells where motors were presumably stalled via the attachment of flagella to the coverslip, reported a value two orders of magnitude faster than our measured . However, this study was performed with stator units fused to a fluorescent protein, which can cause different behaviors from their wild-type counterparts ( 10 , 41 ). The present study reports on the dynamics of wild-type stator units in otherwise unperturbed motors.…”

Does functional specialization of ribosomes really exist?

“…The addition of epitope tags to RPs of interest, while powerful and often necessary, can also suffer from distinct drawbacks. Tagged proteins can have altered function (Swulius and Jensen 2012;Heo et al 2017;Collins et al 2018) or stability (De Marco et al 2004;Kim et al 2013;Natsume et al 2016). These problems are especially acute when dealing with RPs, which are often small and highly charged.…”

GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission

“…However, seldom dissected are the plausible negative structural and functional ramifications of attaching the sizeable, ~ 27 kDa GFP moiety to the protein of interest. Indeed, in many instances, GFP-tagging has been shown to generate unstable fusion products 4 , 5 , cause aberrant protein localization 6 , promote aggregation 7 , prevent assembly 8 , and/or perturb protein function in more than subtle ways 9 – 12 . Such scrutiny has never been imposed on GFP-tagged dynamin-related protein 1 (Drp1), a long-studied yet controversial mechanoenzymatic GTPase recruited from the cytosol to the mitochondrial surface to mediate mitochondrial division 13 , 14 .…”