Ratiometric Oxygen Sensing with H-NOX Protein Conjugates.
Ratiometric Oxygen Sensing with H-NOX Protein Conjugates.
复制标题
使用 H-NOX 蛋白缀合物进行比例氧传感。
DOI：
10.1021/acs.inorgchem.2c01430
复制
发表时间：
2022
影响因子：
4.6
通讯作者：
Marletta,MichaelA
中科院分区：
文献类型：
--
作者：
Lemon,ChristopherM;Hanley,Deirdre;Batka,AllisonE;Marletta,MichaelA
比率传感器是在细胞和组织中起作用的自参考结构，其读数与传感器浓度无关。比率传感的一种策略是利用双色发射，其中一种组分具有依赖于分析物的发射，另一种组分独立于分析物的浓度，作为内标。这样，两组分的强度比就是分析物的定量度量。在这项研究中，利用来自地下嗜热细菌caldanaerobacter的血红素一氧化氮/氧结合蛋白（H-NOX）制备了基于蛋白质的比例氧传感器。天然血红素辅因子被Pd（II）或Pt（II）卟啉取代，作为氧响应磷。进行诱变以在蛋白质表面加入半胱氨酸残基，用于氧不敏感染料的硫醇/马来酰亚胺偶联，该染料作为卟啉的Förster共振能量转移（FRET）供体。虽然基于Pd（II）和Pt（II）的传感器在生物学相关范围内都有响应，但Pd传感器在较低氧浓度下表现出更高的灵敏度。总之，这些传感器代表了一类新的基于蛋白质的比例氧传感器，模块化平台允许为特定应用量身定制氧灵敏度。这项原理验证研究确定了开发和随后改进基于蛋白质的比例氧传感器的关键考虑因素和最佳方法。
Ratiometric sensors are self-referencing constructs that are functional in cells and tissues, and the read-out is independent of sensor concentration. One strategy for ratiometric sensing is to utilize two-color emission, where one component possesses analyte-dependent emission and the other is independent of analyte concentration, serving as an internal standard. In this way, the intensity ratio of the two components is a quantitative measure of the analyte. In this study, protein-based ratiometric oxygen sensors are prepared using the heme nitric oxide/oxygen-binding protein (H-NOX) from the thermophilic bacteriumCaldanaerobacter subterraneus. The native heme cofactor is replaced with a Pd(II) or Pt(II) porphyrin as the oxygen-responsive phosphor. Mutagenesis is performed to incorporate a cysteine residue on the protein surface for thiol/maleimide coupling of the oxygen-insensitive dye, which serves as a Förster resonance energy transfer (FRET) donor for the porphyrin. While both Pd(II)- and Pt(II)-based sensors are responsive over biologically relevant ranges, the Pd sensor exhibits greater sensitivity at lower oxygen concentrations. Together, these sensors represent a new class of protein-based ratiometric oxygen sensors, and the modular platform allows the oxygen sensitivity to be tailored for a specific application. This proof-of-principle study has identified the key considerations and optimal methodologies to develop and subsequently refine protein-based ratiometric oxygen sensors.