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首页> 外文期刊>Biochemistry >Values of Carboxyl Groups in the Native and Denatured States of Barnase: The pK_A Values of the Denatured State Are on Average 0.4 Units Lower Than Those of Model Compounds
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Values of Carboxyl Groups in the Native and Denatured States of Barnase: The pK_A Values of the Denatured State Are on Average 0.4 Units Lower Than Those of Model Compounds

机译：Barnase天然和变性状态中的羧基基团值：变性状态的pK_A值平均比模型化合物低0.4个单位

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摘要

We have determined the pK_A values of the 12 carboxyl residues in the native and denatured state of bamase by a combination of thermodynamic measurements on mutants of charged residues and NMR titration data. The pK_A values of the 11 residues titrating under folding conditions (above pH 2.2) were determined by two-dimensional 'H NMR. The pK_A value of the remaining residue, Asp 93 which forms a salt link with Arg 69 and titrates at much lower pH values, was determined by changes in the pH dependenceof the stability of the protein upon mutation to Asn: pK_A~(Asp93) at low ionic strength (50 mM) and pK_A~(Asp93) at high ionic strength (600 mM). The overall titration of the native state is nonideal, and the piotein retains fractionally ionized residues other than Asp 93 throughout the experimental pH range of 0.2-6,3. Protonation events taking place at pH values below 2 were further characterized by the pH dependence of the unfolding kinetics of wild-type and charge-mutant proteins. By comparing the observed pH dependence of the protein stability with that calculated from the pK_A values for the native protein, we demonstrate that the pK_A values of the denatured state are significantly lower than those reported for model compounds: the pK_A values of the denatured state appear on average 0.4 units lower than previous estimates in the presence of chemical denatutant. The results have direct implications for calculations of the energetics of proton equilibria and suggest that the acid/thermally denatured state is not an extended coil where the residues are isolated from one another by fee intervening solvent but is compact and involves intramolecular charge repulsion.
机译：通过结合带电残基突变体的热力学测量结果和NMR滴定数据，我们确定了bamase天然和变性状态下12个羧基残基的pK_A值。通过二维1 H NMR测定在折叠条件（pH 2.2以上）下滴定的11个残基的pK_A值。通过在突变为Asn时蛋白质稳定性的pH依赖性变化来确定其余残基Asp 93与Arg 69形成盐键并在较低的pH值处滴定的pK_A值：pK_A〜（Asp93）低离子强度（50 mM）和pK_A〜（Asp93），高离子强度（600 mM）。天然状态的总滴定是不理想的，并且在整个实验pH范围0.2-6,3中，叶黄素保留了除Asp 93以外的部分离子化残留物。 pH值低于2时发生的质子化事件进一步通过野生型和电荷突变蛋白的展开动力学的pH依赖性来表征。通过比较观察到的蛋白质稳定性与从天然蛋白质的pK_A值计算得出的pH依赖性，我们证明了变性状态的pK_A值显着低于模型化合物的报告值：出现了变性状态的pK_A值在存在化学变性剂的情况下，平均比以前的估计值低0.4个单位。结果对质子平衡能的计算有直接的影响，并表明酸/热变性状态不是一个扩展的线圈，在该线圈中，残留物通过中间的溶剂相互隔离，但很紧凑，并且涉及分子内电荷排斥。

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