Engineering of the Heme Pocket of an H-NOX Domain for Direct Cyanide Detection and Quantification
Engineering of the Heme Pocket of an H-NOX Domain for Direct Cyanide Detection and Quantification
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DOI：
10.1021/ja101674z
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发表时间：
2010-08-25
影响因子：
15
通讯作者：
Boon, Elizabeth M.
中科院分区：
文献类型：
--
作者：
Dai, Zhou;Boon, Elizabeth M.
已经研究了一种新的氰化物传感系统，即来自腾康热厌氧杆菌 (Tt H-NOX) 的血红素-一氧化氮和/或氧结合域 (H-NOX 域)。通过基于吸光度的简单检测，我们实现了 0.5 μM（类似于 10 ppb）的氰化物检测限，检测范围上限可根据蛋白质浓度进行调节。我们发现多个光谱特征与氰化物浓度呈线性相关。这些光谱特征包括 Soret 带最大值以及光谱的 Soret 和 α/β 带区域中的吸光度变化。与其他氰化物传感方法相比，用于氰化物检测的多个探针使得 Tt H-NOX 传感具有独特性。此外，利用定点诱变，我们合理设计了 Tt H-NOX 的血红素口袋，以改善其氰化物传感特性。使用改变 Tt H-NOX (P115A) 血红素结构的突变体，我们能够引入氰化物的比色检测。通过用更小的残基（缬氨酸，F78V）或更大的残基（酪氨酸，F78Y）取代苯丙氨酸 78，我们证明了与远端口袋空间拥挤和氰化物亲和力的相关性。特别是，F78V Tt H-NOX 显示 CN 结合亲和力和选择性显着增加。因此，我们证明了微调 Tt H-NOX 对氰化物的亲和力和特异性的能力，这表明 Tt H-NOX 可以很容易地定制成实用的氰化物传感器。
A new cyanide sensing system, the Heme-Nitric oxide and/or OXygen binding domain (H-NOX domain) from Thermoanaerobacter tengcongensis (Tt H-NOX), has been investigated. With straightforward absorbance-based detection, we have achieved a cyanide detection limit of 0.5 mu M (similar to 10 ppb) with an upper detection range that is adjustable with protein concentration. We find a linear correlation of multiple spectroscopic features with cyanide concentration. These spectroscopic features include the Soret band maximum and absorbance changes in both the Soret and alpha/beta band regions of the spectrum. Multiple probes for cyanide detection makes sensing with Tt H-NOX unique compared to other cyanide sensing methods. Furthermore, using site-directed mutagenesis, we have rationally engineered the heme pocket of Tt H-NOX to improve its cyanide sensing properties. Using a mutant that alters the heme structure of Tt H-NOX (P115A) we were able to introduce colorimetric detection of cyanide. Through substituting phenylalanine 78 with a smaller (valine, F78V) or a larger residue (tyrosine, F78Y), we demonstrate a correlation with distal pocket steric crowding and affinity for cyanide. In particular, F78V Tt H-NOX shows a significant increase in CN- binding affinity and selectivity. Thus, we demonstrate the ability to fine-tune the affinity and specificity of Tt H-NOX for cyanide, suggesting that Tt H-NOX can be readily tailored into a practical cyanide sensor.