A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications

摘要: The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over last several years, mutagenesis studies have improved folding properties GFP (refs 1,2). However, slow maturation is still big obstacle to use variants visualization. These problems are exacerbated when expressed at 37 degrees C and/or targeted certain organelles. Thus, obtaining that mature more efficiently crucial development expanded research applications. Among variants, yellow proteins (YFPs) relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP be fully stably fluorescent, mutations decrease sensitivity both pH Cl- desired. Here we describe an version named "Venus". Venus contains novel mutation, F46L, which greatly accelerates oxidation chromophore, rate-limiting step maturation. As result other mutations, F64L/M153T/V163A/S175G, folds well tolerant exposure acidosis Cl-. We succeeded in targeting neuropeptide Y-Venus fusion dense-core granules PC12 cells. Its secretion was readily monitored measuring release fluorescence into medium. as acceptor allowed early detection reliable signals resonance energy transfer (FRET) Ca2+ measurements brain slices. With speed efficiency increased resistance environment, will enable labelings were not possible before.