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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A fluorescence resonance energy transfer sensor for the beta -domain of metallothionein.
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A fluorescence resonance energy transfer sensor for the beta -domain of metallothionein.

机译：用于金属硫蛋白的β结构域的荧光共振能量转移传感器。

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摘要

We have designed a nanosensor to study the potential function of metallothionein (MT) in metal transfer and its interactions with redox partners and ligands by attaching two fluorescent probes to recombinant human MT. The specific labeling takes advantage of two different modification reactions. One is based on the fact that recombinant MT has a free N-terminal amino group when produced by the IMPACT T7 expression and purification system, the other on the observation that one human MT isoform (1b) contains an additional cysteine at position 32. It is located in the linker region of the molecule, allowing the introduction of a probe between the two domains. An S32C mutation was introduced into hMT-2. Its thiol reactivity, metal binding capacity, and CD and UV spectra all demonstrate that the additional cysteine contains a free thiol(ate); it perturbs neither the overall structure of the protein nor the formation of the metalthiolate clusters. MT containing only cadmium was labeled stoichiometrically with Alexa 488 succinimidyl ester at the N terminus and with Alexa 546 maleimide at the free thiol group, followed by conversion to MT containing only zinc. Energy transfer between Alexa 488 (donor) and Alexa 546 (acceptor) in double-labeled MT allows the monitoring of metal binding and conformational changes in the N-terminal beta-domain of the protein.
机译：我们设计了一种纳米传感器，通过将两个荧光探针连接到重组人MT上，研究金属硫蛋白（MT）在金属转移中的潜在功能及其与氧化还原伙伴和配体的相互作用。特定标记利用了两个不同的修饰反应。一种基于这样的事实，即重组MT在通过IMPACT T7表达和纯化系统产生时具有一个游离的N端氨基，另一种基于观察到一个人MT同工型（1b）在32位含有一个额外的半胱氨酸。 cDNA位于分子的连接区中，从而允许在两个结构域之间引入探针。 S32C突变被引入hMT-2。它的硫醇反应性，金属结合能力以及CD和UV光谱均表明，另外的半胱氨酸含有游离的硫醇（酸酯）。它既不会干扰蛋白质的整体结构，也不会干扰金属硫醇盐簇的形成。仅在化学计量上用N末端的Alexa 488琥珀酰亚胺酯和游离硫醇基上的Alexa 546马来酰亚胺标记仅含镉的MT，然后转化为仅含锌的MT。双标记MT中Alexa 488（供体）和Alexa 546（受体）之间的能量转移可监测蛋白质N端β结构域中的金属结合和构象变化。

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