Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin
Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin
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DOI：
10.1073/pnas.0600774103
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发表时间：
2006-05-09
影响因子：
11.1
通讯作者：
Dennison, Christopher
中科院分区：
文献类型：
--
作者：
Chan Li;Yanagisawa, Sachiko;Dennison, Christopher
铜结合蛋白天青蛋白（一种铜氧还蛋白）的主要活性位点环已从C（112）TFPGH（117）SALM（121）缩短为（CTPHPM 118）-T-112-P-115（来自铜氧还蛋白amicyanin的天然环），也缩短为（CTPHPM 117）-T-112-P-115。Cu（II）位点结构几乎不受缩短的影响，在具有（CTPHPM 117）-T-112-P-115环序列的变体中，Cu（I）中心在碱性pH下的结构也是如此。微妙的光谱差异，由于在Cu（II）网站的自旋密度分布的改变，可以主要归因于氢键模式的变化。电子转移几乎不受引入（CTPHPFM 118）-T-112-P-115环的影响，但去除Phe残基对反应性有相当大的影响，可能是因为减少了同二聚体的形成。在温和的酸性pH值下，His-115配体质子化并从亚铜离子解离，这种作用对铜氧还蛋白的反应性具有显著影响。这些研究表明，当引入天青蛋白时，amicyanin环采用与天然蛋白中发现的构象相同的构象，比天然存在的C末端活性位点环短的C末端活性位点环可以支持功能性T1铜位点，CTPHPM是结合这种普遍存在的电子传递中心所需的最小环长度，并且金属结合环的长度和序列调节金属蛋白活性位点的一系列结构和功能特征。
The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C(112)TFPGH(117)SALM(121) to (CTPHPFM118)-T-112-P-115 (the native loop from the cupredoxin amicyanin) and also to (CTPHPM117)-T-112-P-115. The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the (CTPHPM117)-T-112-P-115 loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the (CTPHPFM118)-T-112-P-115 loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.