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.

bias-dependent asymmetry in the speed attained in the two rotation directions. 16All these effects could be mitigated by the insertion of a linker at the fusion point.

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.Fusion of a fluorescent protein to a protein of interest is an invaluable tool for the study of protein function in a broad, and still expanding range of in vivo and in vitro systems. However, it is widely recognized that the presence of fluorescent proteins can alter functional properties of the native protein [1][2][3][4][5] . Careful assessment of these side effects and strategies to minimize them have therefore been critical to the success of fluorescent protein fusions (FPFs) [6][7][8][9] . The continuing development of FPF-based approaches to explore novel phenomena also calls for new insight into the associated side effects and methods to alleviate them [10][11][12] . One direction of research for which this holds is the study of the internal dynamics of proteins complexes 13,14 . Recently, several studies have made use of FPFs to study the internal subunit dynamics of the bacterial flagellar motor (BFM) [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] . Although FPFs were shown to affect BFM function by decreasing the chemotactic motility of cells and average speed of motors 15 , a quantitative characterization of the impact on the BFM mechanical behavior is lacking. Such perturbations limit the depth to which the internal dynamics and function of the BFM, and protein complexes in general, can be probed with FPFs. Linker peptides inserted at the fusion point have been instrumental in minimizing non-native behaviour of FPFs in many biological systems [8][9][10][11] , but their use in the study of protein-complex dynamics is limited in the BFM has thus far been limited.The BFM is located at the base of each flagellum in the membrane of many motile bacteria (Fig. 1A). The torque gene...