Kinetic Studies of Protein Folding Using Protein Engineering

本研究以大肠杆菌质粒克隆表达的葡萄球菌核酸酶和山羊α-乳白蛋白为研究对象，探讨其蛋白质折叠机制。使用这些蛋白质的各种定点突变体，熔融球状态的稳定性和复性动力学进行了分析。获得了以下结果。(1)为了研究脯氨酸异构化对核酸酶复性动力学的影响，构建了两个脯氨酸突变体Pro 117 Gly和Pro56 Ala。(2)已知核酸酶的突变体分为两类：I类突变体稳定熔融球状态，II类突变体使熔融球状态不稳定。具有I类（Val 66 Leu、Gly 88 Val和它们的双突变体）和II类（Ala 90 Ser、Ala 69 Thr和它们的双突变体）突变的蛋白质从具有相应突变质粒的大肠杆菌中纯化。每个突变体蛋白质的解折叠转变通过在肽区域中的CD光谱测量。正如预期的那样，I类突变体的二级结构比野生型蛋白更稳定，而II类突变体的二级结构不太稳定。(3)改变分子核心疏水性的突变（Ala 30 Ile、Ala 30 Thr和Thr 33 Ile）已被引入α-乳白蛋白中，并且通过CD光谱研究了突变对熔融球状态的稳定性的影响。增加核心疏水性的Ala 30 Ile和Thr 33 Ile突变增加熔融球的稳定性，而降低疏水性的Ala 30 Thr突变降低熔融球的稳定性。

In this study, staphylococcal nuclease and goat alpha-lactalbumin cloned and expressed by plasmids of Escherichia coli were used to investigate the mechanism of protein folding. Using various site-directed mutants of these proteins, stability of the molten globule state and kinetics of refolding were analyzed. The following results were obtained.(1) In order to investigate effect of proline isomerization on the refolding kinetics, two proline mutants, Pro117Gly and Pro56Ala, of nuclease were constructed. (2) The mutants of nuclease are known to be classified into two classes : Class I mutants stabilize and class II mutants destabilize the molten globule state. The proteins with the class I (Val66Leu, Gly88Val and their double mutant) and class II (Ala90Ser, Ala69Thr and their double mutant) mutations were purified from Escherichia coli that has the respective mutant plasmids. The unfolding transition of each mutant protein was measured by CD spectroscopy in the peptide region. As expected, the secondary structure of the class I mutants is more stable than that of the wild-type protein, while that of the class II mutants is less stable. (3) The mutations (Ala30Ile, Ala30Thr and Thr33Ile) that change the hydrophobicity of the core of the molecule have been introduced in alpha-lactalbumin, and the effect of the mutations on the stability of the molten globule state was investigated by CD spectroscopy. The Ala30Ile and Thr33Ile mutations that increase the core hydrophobicity increase the stability of the molten globule, while the Ala30Thr mutation that decreases the hydrophobicity decreases the stability of the molten globule.