Lohman Jeremy R, Remington S James
Institute of Molecular Biology and Departments of Chemistry and Physics, University of Oregon, Eugene, Oregon 97403, USA.
Biochemistry. 2008 Aug 19;47(33):8678-88. doi: 10.1021/bi800498g. Epub 2008 Jul 25.
Green fluorescent protein (GFP) indicators were previously developed that rapidly and quantitatively respond to changes in the thiol/disulfide equilibrium within subcellular compartments. In these indicators, surface-exposed cysteines residues were introduced so as to form a labile redox-active disulfide that in turn controls the emission properties of the internal chromophore. The biosensors have been shown to be effective reporters of the thiol/disulfide status within reducing compartments such as the mitochondria and cytosol for several cell types. However, due to the high thermodynamic stability of the introduced disulfide bond, the indicators are not useful for quantitative analysis within more oxidizing compartments such as the endoplasmic reticulum. Here we report the development of a new family of GFP-based redox indicators (roGFP1-iX) in which the thermodynamic stability of the disulfide is substantially lowered by insertion of a single amino acid into the main chain, adjacent to cysteine 147. The insertions result in indicators with midpoint potentials of -229 to -246 mV and are thus better suited for study of relatively oxidizing subcellular compartments. Atomic resolution crystallographic analyses suggest that two important factors act to destabilize the disulfide linkage in roGFP1-iX. In the oxidized state, an unusual non-proline cis-peptide bond adjacent to one of the cysteines introduces geometric strain into the system, while in the reduced state, a dramatic loop opening lowers the effective concentration of the reacting species.
绿色荧光蛋白（GFP）指示剂此前已被开发出来，它能快速且定量地响应亚细胞区室中硫醇/二硫键平衡的变化。在这些指示剂中，引入了暴露于表面的半胱氨酸残基，以形成不稳定的氧化还原活性二硫键，进而控制内部发色团的发射特性。对于几种细胞类型而言，这些生物传感器已被证明是还原区室（如线粒体和细胞质）中硫醇/二硫键状态的有效报告分子。然而，由于引入的二硫键具有很高的热力学稳定性，这些指示剂对于在内质网等氧化性更强的区室中进行定量分析并无用处。在此，我们报告了一种基于GFP的新型氧化还原指示剂（roGFP1-iX）家族的开发情况，其中通过在主链中紧邻半胱氨酸147插入单个氨基酸，二硫键的热力学稳定性大幅降低。这些插入操作产生了中点电位为-229至-246 mV的指示剂，因此更适合用于研究相对氧化性的亚细胞区室。原子分辨率晶体学分析表明，有两个重要因素致使roGFP1-iX中的二硫键不稳定。在氧化状态下，与其中一个半胱氨酸相邻的异常非脯氨酸顺式肽键会给系统引入几何应变，而在还原状态下，一个显著的环打开会降低反应物种的有效浓度。
