Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)
Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)
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DOI：
10.1073/pnas.1618087114
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发表时间：
2017-03-14
影响因子：
11.1
通讯作者：
Boxer, Steven G.
中科院分区：
文献类型：
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作者：
Lin, Chi-Yun;Both, Johan;Boxer, Steven G.
通过将GFP表达为连接至仅在彼此互补时发荧光的单独蛋白质的两个或更多个组成部分，分裂GFP已被广泛应用于监测蛋白质-蛋白质相互作用。虽然这种互补通常是不可逆的，但先前已经表明，光加速了非共价连接的β链从环状排列的分裂GFP的解离，使得相互作用是可逆的。可逆互补是期望的，但是光解离具有太低的效率（量子产率< 1%）而不能用作光遗传学工具。了解这种低效率的物理起源可以提供策略，以改善it. We阐明链光解的机制，通过测量其速率对光强和点突变的依赖性。结果表明，链的光解离是一个两步过程，包括光激活的顺反异构化的发色团，然后光无关的链解离。然后利用速率对温度的依赖关系建立了沿着光解反应坐标的势能面图。由此产生的能量函数模型揭示了速率限制过程是从电子激发态的过渡到基态PES伴随顺反异构化。将分裂GFP与光敏黄蛋白和视紫红质等光敏蛋白进行比较，为提高光解离量子产率提供了潜在的策略。
Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached beta-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield < 1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.