LeMaster David M, Anderson Janet S, Hernández Griselda
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York 12201, USA.
Biochemistry. 2006 Aug 22;45(33):9956-63. doi: 10.1021/bi0605540.
Amide exchange rates were measured for Pyrococcus 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 A, 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 in Pf rubredoxin 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.
对用Zn(II)、Ga(III)或Ge(IV)取代的嗜热栖热菌（Pf）红素氧还蛋白测定了酰胺交换速率。对于活性位点金属周围高度受保护的酰胺，碱催化的交换速率常数每单位电荷增加高达3000倍，在比较Zn(II)和Ge(IV)取代的蛋白质时，产生超过8 kcal/mol的明显残基特异性构象能量降低。然而，该蛋白质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射线结构中酰胺氮与金属之间的距离成反比，至少可达12 Å的距离，这与酰胺氮pK的静电势依赖性移动一致。这种强烈相关的距离依赖性与交换活性构象的类似天然结构一致。红素氧还蛋白结构内部的电场电势导致单个酰胺氢的交换活性状态的速率变化高达一百万倍。然而，Pf红素氧还蛋白中这些静电相互作用的强度似乎与先前在其他蛋白质中报道的强度相当。因此，与氢交换数据的传统分析相反，对于通过非全局转变发生的交换过程，残余构象结构通常会调节观察到的速率。虽然这必然会使这些交换活性状态的构象平衡的估计复杂化，但这种对残余结构的依赖性可以提供对这些瞬态状态构象的洞察。
