Aromatic thiol pKa effects on the folding rate of a disulfide containing protein
Aromatic thiol pKa effects on the folding rate of a disulfide containing protein
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DOI：
10.1021/bi034305c
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发表时间：
2003-10-14
期刊：
影响因子：
2.9
通讯作者：
Lees, WJ
中科院分区：
文献类型：
--
作者：
Gough, JD;Gargano, JM;Lees, WJ
通过重组DNA技术生产蛋白质通常需要体外折叠包涵体，其是蛋白质聚集体。为了产生用于含二硫化物的蛋白质的体外折叠的更有效的氧化还原缓冲液，研究了芳香族硫醇增加乱序RNA酶A的折叠速率的能力。乱序RNA酶A是完全氧化的RNA酶A，具有相对随机分布的二硫键。通过分析pK（a）值范围为5.2至6.6的五种对位取代芳香族硫醇，研究了硫醇pK（a）值的重要性。在pH 6.0下测量折叠，其中硫醇的pK（a）值将高于、低于或等于溶液pH。因此，硫醇和硫醇盐的相对浓度将在整个系列中变化。在pH 6.0时，芳香族硫醇使RNase A的折叠速率比标准添加剂谷胱甘肽所观察到的折叠速率增加10-23倍。在最佳条件下，表观速率常数随巯基pK（a）值的减小而增大。当溶液中质子化硫醇的浓度约为2 mM时，出现最佳条件，尽管总硫醇浓度变化很大。溶液中质子化硫醇浓度的重要性可以基于平衡效应来理解。动力学研究表明，氧化还原缓冲液作为亲核试剂和/或中心硫醇参与决定蛋白质折叠过程中发生的硫醇二硫键交换反应的关键速率。芳香族硫醇被证明是动力学更快，更通用的比经典的脂肪族硫醇氧化还原缓冲液。
The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.