Re-engineering redox-sensitive green fluorescent protein for improved response rate
Re-engineering redox-sensitive green fluorescent protein for improved response rate
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DOI：
10.1110/ps.051734306
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发表时间：
2006-01-01
期刊：
影响因子：
8
通讯作者：
Remington, SJ
中科院分区：
文献类型：
--
作者：
Cannon, MB;Remington, SJ
绿色荧光蛋白的氧化还原敏感变异体(RoGFP)已经被开发出来，可以通过荧光激发比率法“实时”监测细胞隔间的氧化还原状态。然而，这些探针的反应时间限制了对某些快速氧化事件的研究，例如细胞信号中的过氧化氢爆发。在roGFPI(变种命名为roGFPI-RI至-R14)中，引入的二硫键附近最多三个带正电的氨基酸的替代大大提高了应答率。测定了H_2O_2氧化、DTT还原和氧化还原中点电位的准一级速率常数。随着每增加一个带正电荷的取代，速率常数几乎翻了一番，达到几乎全部数量级。中点电位与速率的增加高度相关，随着正取代数的增加而变得更具氧化性。测定了具有相反二硫化物氧化态的两个变体的晶体结构：含有两个碱性取代基的氧化“R7”的2.2埃分辨结构。和一个碱性和一个酸性取代的还原“R8”的1.95埃分辨率结构。用非线性Poisson-Boltzrriann(PB)计算精确预测了取代基对反应速率常数的影响。讨论了取代基对二聚体形成、相对氧化中点电位、氧化还原速率的影响。结果表明，RoGFP是定量分析硫醇转移反应影响因素的良好模型体系。由于反应速度和等电点的显著提高，roGFP1-R12最适合用于活细胞。
Redox-sensitive variants of the green fluorescent protein (roGFPs) had previously been developed that allow "real-time" monitoring of the redox status of cellular compartments by fluorescence excitation ratiometry. However, the response time of these probes limits the study of certain rapid oxidative events, such as H2O2 bursts in cell signaling. The substitution of up to three positively charged amino acids adjacent to the introduced disulfide in roGFPI (variants designated roGFPI-RI through -R14) substantially improved the response rate. The pseudo First-order rate constants for oxidation by H2O2 and reduction by DTT and redox midpoint potentials were determined. The rate constants approximately doubled with each additional positively charged substitution, to nearly ail order of magnitude total. The midpoint potentials are highly correlated with the rate increases, becoming more oxidizing with increasing numbers of positive substitutions. Crystal structures of two variants with opposite disulfide oxidation states have been determined: a 2.2 angstrom resolution structure of oxidized "R7" containing two basic substitutions. and a 1.95 angstrom resolution structure of reduced "R8" with one basic and one acidic substitution. Nonlinear Poisson-Boltzrriann (PB) calculations are shown to accurately predict the effects of the substitutions on the rate constants. The effects of the substitutions on dimer formation, relative oxidative midpoint potentials, and oxidation and reduction rates are discussed. roGFPs are demonstrated to Constitute an excellent model system for quantitative analysis of factors influencing thiol transfer reactions. roGFP1-R12 is most suitable for use in live cells, due to significantly increased reaction rate and increased pI.