Carboxyl pKa Values and Acid Denaturation of BBL
Carboxyl pKa Values and Acid Denaturation of BBL
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DOI：
10.1016/j.jmb.2010.08.052
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发表时间：
2010-10-22
影响因子：
5.6
通讯作者：
Fersht, Alan R.
中科院分区：
文献类型：
--
作者：
Arbely, Eyal;Rutherford, Trevor J.;Fersht, Alan R.
蛋白质BBL在pH 1.2和8.0之间经历结构转变和酸变性。使用NMR光谱法，我们测量了该pH范围内所有羧基残基的pK（a）值。我们采用C-13直接检测二维IPAP（同相反相）CACO NMR光谱监测不同羧基的电离状态，并证明了其优于其他NMR技术在测量羧酸残基的pKa值。两个残基Glu 161和Asp 162具有显著降低的pKa值，表明这些残基与His 166一样参与稳定静电相互作用的网络。其他羧酸盐具有未扰动值。变性自由能的pH依赖性定量描述了这三个残基的扰动pKa的电离，并使用热力学循环，我们可以计算它们在天然和变性状态的pK（a）s以及不同质子化状态的变性平衡常数。我们还测量了C-13（α）的化学位移的个别残基作为pH值的函数。这些变化感测结构转变，而不是电离，他们滴定与pH值的变性平衡常数的变化一致。BBL E161 Q折叠的动力学测量表明，在pH 7时，与Glu 161的稳定相互作用主要形成于过渡态。我们还发现BBL在酸变性状态下仍然存在局部相互作用，这在一定程度上减弱了酸变性状态的柔性。(C)2010爱思唯尔有限公司版权所有。
The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed C-13 direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pKa values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pKa values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pKa, and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured C-13(alpha) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state. (C) 2010 Elsevier Ltd. All rights reserved.