Copper sensing based on the far-red fluorescent protein, HcRed, from Heteractis crispa
Copper sensing based on the far-red fluorescent protein, HcRed, from Heteractis crispa
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DOI：
10.1016/j.ab.2007.05.018
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发表时间：
2007-11-01
影响因子：
2.9
通讯作者：
Deo, Sapna K.
中科院分区：
文献类型：
--
作者：
Rahimi, Yasmeen;Shrestha, Suresh;Deo, Sapna K.
在这篇文章中，我们报告的铜（Cu 2+）的远红荧光蛋白，HcRed的结合特性的第一次，和它的应用在开发一个无试剂传感系统的铜。其中反向细胞荧光低的HcRed = 645 nm的远红光发射应证明在传感系统的开发中是有利的。在我们实验室进行的研究中，我们发现HcRed的荧光在铜离子（Cu 2+）的存在下被淬灭。通过在铜的存在和不存在下产生的UV-可见光和圆二色性光谱以及在不同温度下的Stern-Volmer图获得的结果表明，在铜的存在下HcRed荧光的静态淬灭，可能是通过形成铜-蛋白质复合物。在此基础上，我们开发了一种基于赫雷德作为生物传感元件的无试剂传感系统，用于检测铜（II）。获得了在纳摩尔范围内的Cu 2+的检测限。发现HcRed与其他二价离子相比选择性地结合铜离子。观察到铜结合的解离常数为3.6 μ M。组氨酸和半胱氨酸残基通常参与蛋白质内的铜结合;因此，研究这些氨基酸在HcRed中的作用。我们分别用碘乙酰胺和焦碳酸二乙酯化学修饰Cys和His残基。研究了铜的加入对化学修饰的HcRed荧光的影响。赫雷德的His修饰显著影响铜离子结合，指出组氨酸可能是参与赫雷德中铜离子结合的氨基酸残基。还开发了一种基于铜固定化亲和柱的HcRed纯化策略，该柱在蛋白质上不添加任何亲和标签。基于HcRed的铜传感系统可以潜在地用于通过遗传编码生物传感元件来执行细胞内铜检测，或者可以用于环境传感。(c)2007爱思唯尔公司All rights reserved.
In this article, we report for the first time on the copper (Cu2+) binding characteristics of the far-red fluorescent protein, HcRed, and its application in the development of a reagentless sensing system for copper. The far-red emission of HcRed = 645 nm) where back-around cellular fluorescence is low should prove to be advantageous in the development of the sensing system. In the studies performed in our laboratory, we found that the fluorescence of HcRed is quenched in the presence of copper ions (Cu2+ ). The results obtained through UV-visible and circular dichroism spectra generated in the presence and absence of copper, as well as Stern-Volmer plots at different temperatures, indicate static quenching of HcRed fluorescence in the presence of copper, possibly through the formation of a copper-protein complex. On the basis of this observation, we developed a reagentless sensing system for the detection of copper(II) based on HeRed as the biosensing element. A detection limit for Cu2+ in the nanomolar range was obtained. HcRed was found to bind copper ions selectively when compared with other divalent ions. A dissociation constant of 3.6 mu M was observed for copper binding. Histidine and cysteine residues are commonly involved in copper binding within proteins; therefore, to investigate the role of these amino acids present in HcRed. we chemically modified Cys and His residues using iodoacetamide and diethyl pyrocarbonate, respectively. The effect of copper addition on the fluorescence of the chemically modified HcRed was investigated. The His modification of HeRed substantially affected copper ion binding, pointing to histidine as the possible amino acid residue involved in the binding of copper ions in HeRed. A purification strategy for HcRed was also developed based on a copper immobilized affinity column without the addition of any affinity tag on the protein. The HcRed-based copper sensing system can potentially be employed to perform intracellular copper detection by genetically encoding the biosensing element or can be employed in environmental sensing. (c) 2007 Elsevier Inc. All rights reserved.