New findings on GFP-like protein application as fluorescent tags: Fibrillogenesis, oligomerization, and amorphous aggregation
2021
Stepanenko, Olga V. | Sulatsky, Maksim I. | Mikhailova, Ekaterina V. | Kuznetsova, Irina M. | Turoverov, Konstantin K. | Stepanenko, Olesya V. | Sulatskaya, Anna I.
Green fluorescent proteins (GFP) are commonly used as fluorescent tags and biosensors in cell biology and medicine. However, the propensity of GFP-like proteins to aggregate and the consequence of intermolecular interaction for their application have not been thoroughly examined. In this work, alternative aggregation pathways of superfolder green fluorescent protein (sfGFP) were demonstrated using a spectroscopic and microscopic study of the samples prepared by equilibrium microdialysis. Besides oligomerization of native monomers, we showed for the first time the condition-specific formation by sfGFP of either amyloid fibrils (at increased temperature or acidity) or amorphous aggregates (at physiological conditions). Both types of sfGFP aggregates had lost green fluorescence and were toxic to cells. Thus, when using GFP-like proteins as fluorescent tags, one should take into account their high ability to form aggregates with lost unique visible fluorescence in the cellular environment, which affects cell viability. Moreover, the results of this work cast doubt on the correctness of the data on the fibrillogenesis of various amyloidogenic proteins obtained using their fusion with GFP-like proteins.
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