Role of native-state structure in rubredoxin native-state hydrogen exchange.
Role of native-state structure in rubredoxin native-state hydrogen exchange.
复制标题
天然态结构在红氧还蛋白天然态氢交换中的作用。
DOI：
10.1021/bi0605540
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Hernandez,Griselda
中科院分区：
文献类型：
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作者：
LeMaster,DavidM;Anderson,JanetS;Hernandez,Griselda
测定了用Zn（II）、Ga（III）或Ge（IV）取代furiosus(Pf) rubredoxin的酰胺交换率。碱基催化的交换速率常数每单位电荷增加高达3000倍的高度保护酰胺周围的活性位点金属，产生明显的残基特异性构象能降低超过8千卡/摩尔在Zn(II)-和Ge(IV)-取代的蛋白质比较。然而，蛋白质的许多其他酰胺的交换动力学对这些金属取代不敏感。这些差异速率与x射线结构中酰胺氮和金属之间的距离呈负相关，距离至少为12 Å，与酰胺氮pK的静电电位依赖位移一致。这种强相关的距离依赖与交换能力构象的天然结构一致。红氧还蛋白结构内部的电场电位使单个酰胺氢交换态的速率变化多达一百万倍。然而，pfrubredoxin中这些静电相互作用的强度似乎与先前报道的其他蛋白质中的静电相互作用相当。因此，与传统的氢交换数据分析相反，对于通过非全局转变发生的交换过程，残留的构象结构通常会调节观察到的速率。虽然这必然使这些交换态的构象平衡的估计复杂化，但这种对剩余结构的依赖可以提供对这些瞬态构象的洞察。
Amide exchange rates were measured forPyrococcus furiosus(Pf) rubredoxin substituted with either Zn(II), Ga(III), or Ge(IV). Base-catalyzed exchange rate constants increase up to 3000-fold per unit charge for the highly protected amides surrounding the active site metal, yielding apparent residue-specific conformational energy decreases of more than 8 kcal/mol in a comparison of the Zn(II)- and Ge(IV)-substituted proteins. However, the exchange kinetics for many of the other amides of the protein are insensitive to these metal substitutions. These differential rates are inversely correlated with the distance between the amide nitrogen and the metal in the X-ray structure, out to a distance of at least 12 Å, consistent with an electrostatic potential-dependent shifting of the amide nitrogen pK. This strongly correlated distance dependence is consistent with a nativelike structure for the exchange-competent conformations. The electric field potential within the interior of the rubredoxin structure gives rise to a change of as much as a million-fold in the rate for the exchange-competent state of the individual amide hydrogens. Nevertheless, the strength of these electrostatic interactions inPfrubredoxin appears to be comparable to those previously reported within other proteins. As a result, contrary to the conventional analysis of hydrogen exchange data, for exchange processes that occur via nonglobal transitions, the residual conformational structure will often modulate the observed rates. Although this necessarily complicates the estimation of the conformational equilibria of these exchange-competent states, this dependence on residual structure can provide insight into the conformation of these transient states.