Rodriguez Erik A, Tran Geraldine N, Gross Larry A, Crisp Jessica L, Shu Xiaokun, Lin John Y, Tsien Roger Y
Department of Pharmacology, University of California, San Diego, La Jolla, California, USA.
School of Medicine, University of California, San Francisco, San Francisco, California, USA.
Nat Methods. 2016 Sep;13(9):763-9. doi: 10.1038/nmeth.3935. Epub 2016 Aug 1.
Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.
远红荧光蛋白(FPs)适用于体内成像,因为与青色、绿色、黄色和橙色荧光蛋白相比,这些分子被内源性生物分子散射、吸收或重新发射的光更少。我们从别藻蓝蛋白α亚基(APCα)开发了一类新的荧光蛋白。天然的别藻蓝蛋白需要一种裂合酶来结合藻蓝胆素。我们将进化后的荧光蛋白命名为小型超红荧光蛋白(smURFP),它无需裂合酶就能共价连接胆绿素(BV)发色团,激发发射峰为642/670纳米,具有较大的消光系数(180,000 M(-1)cm(-1))和量子产率(18%),其光稳定性与增强型绿色荧光蛋白(eGFP)相当。与细菌光敏色素(BPH)荧光蛋白相比,smURFP的BV掺入率和蛋白质稳定性显著更高。此外,BV的供应受膜通透性限制,而smURFP(但不是BPH荧光蛋白)可以掺入一种膜通透性更高的BV类似物,使得smURFP的荧光与水母或珊瑚荧光蛋白相当。利用smURFP和一种BPH荧光蛋白构建了一种远红和近红外荧光细胞周期指示剂。