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首页> 外文期刊>Journal of Molecular Biology >Carboxyl pK(a) values and acid denaturation of BBL.
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Carboxyl pK(a) values and acid denaturation of BBL.

机译：BBL的羧基pK（a）值和酸变性。

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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 (13)C 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 pK(a) values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK(a) 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 pK(a), 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 (13)C(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.
机译：BBL蛋白在pH 1.2和8.0之间经历结构转变和酸变性。使用NMR光谱，我们测量了该pH范围内所有羧基残基的pK（a）值。我们采用（13）C直接检测二维IPAP（同相反相）CACO NMR光谱法来监测不同羧基的电离状态，并证明了它在测量羧基残基的pK（a）值方面优于其他NMR技术。两个残基Glu161和Asp162的pK（a）值明显降低，表明这些残基与His166一样都参与了稳定静电相互作用的网络。其他羧酸盐的值不受干扰。通过扰动的pK（a）的这三个残基的电离来定量描述变性自由能的pH依赖性，并且使用热力学循环，我们可以计算它们在天然和变性状态下的pK（a）s以及不同质子化态变性的平衡常数。我们还测量了各个残基的（13）Cα化学位移与pH的关系。这些位移感知的是结构转变而非电离，它们的pH值与变性平衡常数的变化相符。 BBL E161Q折叠的动力学测量表明，在pH值为7时，与Glu161的稳定相互作用主要在过渡态下形成。我们还发现，在BBL的酸变性状态下，局部相互作用仍然存在，这在一定程度上削弱了酸变性状态的柔韧性。

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