分类： 生物学 >> 生物物理学 提交时间： 2016-05-18
Liu, Xiaohong
Jiang, Li
Li, Jiasong
Wang, Li
Zhou, Qing
Lv, Xiaoxuan
Gong, Weimin
Wang, Jiangyun
Lu, Yi
Lu, Yi
Yu, Yang
摘要： Photo-induced electron transfer (PET) is ubiquitous for photosynthesis and fluorescent sensor design. However, genetically coded PET sensors are underdeveloped, due to the lack of methods to site-specifically install PET probes on proteins. Here we describe a family of acid and Mn(III) turn-on fluorescent protein (FP) sensors, named iLovU, based on PET and the genetic incorporation of superior PET quenchers in the fluorescent flavoprotein iLov. Using the iLovU PET sensors, we monitored the cytoplasmic acidification process, and achieved Mn(III) fluorescence sensing for the first time. The iLovU sensors should be applicable for studying pH changes in living cells, monitoring biogentic Mn(III) in the environment, and screening for efficient manganese peroxidase, which is highly desirable for lignin degradation and biomass conversion. Our work establishes a platform for many more protein PET sensors, facilitates the de novo design of metalloenzymes harboring redox active residues, and expands our ability to probe protein conformational dynamics.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-12
Lv, Xiaoxuan
Li, Jiasong
Zhou, Qing
Wang, Jiangyun
Lv, Xiaoxuan
Li, Jiasong
Zhou, Qing
Wang, Jiangyun
Yu, Yang
Lu, Yi
Cui, Chang
Mukherjee, Arnab
Lu, Yi
Hosseinzadeh, Parisa
Lu, Yi
Nilges, Mark J.
摘要： While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pK(a) and reduction potential in O-2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O-2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O-2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-11
Lv, Xiaoxuan
Hu, Cheng
Gao, Feng
Li, Jiasong
Liu, Xiaohong
Deng, Kai
Zheng, Peng
Gong, Weimin
Wang, Jiangyun
Lv, Xiaoxuan
Hu, Cheng
Gao, Feng
Li, Jiasong
Liu, Xiaohong
Deng, Kai
Zheng, Peng
Gong, Weimin
Wang, Jiangyun
Zhou, Meng
Xia, Andong
摘要： Electron transfer (ET) is widely used for driving the processes that underlie the chemistry of life. However, our abilities to probe electron transfer mechanisms in proteins and design redox enzymes are limited, due to :the lack of methods to site-specifically insert electron acceptors into proteins in vivo. Here we describe the synthesis and genetic incorporation of 4-fluoro-3-nitrophenylalanine (FNO(2)Phe), which has similar reduction potentials to NAD(P)H and ferredoxin, the most important biological reductants. Through the genetic incorporation of FNO2Phe into green fluorescent:protein (GFP) and femtosecond transient absorption measurement, we show that photoinduced electron transfer (PET) from the GFP chromophore to FNO2Phe occurs very fast (within 11 ps), which is comparable to that of the first electron transfer step in photosystem I, from P700* to A(0). This genetically encoded, low-reduction potential unnatural amino acid (UAA) can significantly in-Trove our ability to investigate electron transfer mechanisms in complex reductases and facilitate the design of miniature proteins that mimic their functions.
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