Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme.
Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme.
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DOI：
10.1021/jacs.1c12305
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发表时间：
2022-03-09
影响因子：
15
通讯作者：
Boxer, Steven G.
中科院分区：
文献类型：
--
作者：
Lin, Chi-Yun;Romei, Matthew G.;Mathews, Irimpan I.;Boxer, Steven G.
过去的几十年见证了全新蛋白质设计的爆炸式增长，其支架的范围非常广泛。然而，设计出能够与自然存在的催化剂以及仿生或非生物催化剂竞争的催化功能仍然具有挑战性。尽管定向进化经常提供有效的解决方案，但健康状况仍然不明朗。绿色荧光蛋白（GFP）是重新设计最多的蛋白质之一，它已经彻底改变了生物成像和分析。虽然不是传统意义上的酶，但gfp具有与传统基态催化剂相同的空间和静电起源的竞争激发态衰变途径，并且它们通过相同的原理对多种反应结果施加精细的控制。因此，GFP是一种“激发态酶”。本文表明，合理设计包含环境突变和取代发色团的突变体和杂交种，为描述空间位阻和静电对gfp激发态催化的影响的定量模型和预测提供了基础。由于这两种扰动都可以选择性地偏置光异构化途径，因此可以预测并证明具有荧光量子产率（FQYs）和为特定应用定制的光电开关特性的gfp。通过光谱学很容易获得gfp的潜在能量景观，为蛋白质功能设计提供了一个重要的缺失环节，可推广到催化剂设计。
The past decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or nonbiological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most redesigned proteins. While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional ground-state catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an “excited-state enzyme”. Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs. As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics tailored for specific applications could be predicted and then demonstrated. The underlying energetic landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.
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影响因子：
16.6
作者：
Fried SD;Boxer SG
通讯作者：
Boxer SG
影响因子：
15
作者：
Kotzler, Miriam P.;Robinson, Kyle;Withers, Stephen G.
通讯作者：
Withers, Stephen G.
DOI：
10.1073/pnas.1618087114
发表时间：
2017-03-14
影响因子：
11.1
作者：
Lin, Chi-Yun;Both, Johan;Boxer, Steven G.
通讯作者：
Boxer, Steven G.
影响因子：
4.6
作者：
Drobizhev, Mikhail;Hughes, Thomas E.;Rebane, Aleksander
通讯作者：
Rebane, Aleksander
影响因子：
48
作者：
Alley, Ethan C.;Khimulya, Grigory;Church, George M.
通讯作者：
Church, George M.