Reversibility and hierarchy of thermal transition of hen egg-white lysozyme studied by small-angle X-ray scattering
Reversibility and hierarchy of thermal transition of hen egg-white lysozyme studied by small-angle X-ray scattering
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DOI：
10.1016/s0006-3495(99)77374-1
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发表时间：
1999-04-01
影响因子：
3.4
通讯作者：
Hirai, M
中科院分区：
文献类型：
--
作者：
Arai, S;Hirai, M
为了阐明蛋白质折叠和去折叠的机制，在低蛋白质浓度下进行了许多蛋白质热变性的研究，因为在许多情况下，热变性伴随着很大的聚集趋势。由于小角X射线散射（SAXS）测量易于使用低浓度的蛋白质溶液，以避免聚集，SAXS已被认为是非常难以观察到的热结构转变的详细特征，如分子内的结构变化。通过使用同步辐射SAXS，我们已经发现，蛋白质之间的排斥粒子间的相互作用的存在下，可以保持溶质颗粒分开，以防止在热变性过程中的进一步聚集，并且在这样的条件下，鸡蛋白溶菌酶（HEWL）的热结构转变保持高的可逆性，即使在5%w/v HEWL低于pH值类似于5。由于使用了高浓度的溶液，散射数据具有足够高的统计精度来讨论依赖于结构等级的热结构转变。因此，HEWL的三级结构变化主要从由差示扫描量热法测量确定的起始温度开始，这伴随着大量的热吸收，而对应于结构域间相关性和多肽链排列的分子内结构变化在上述主要转变之前开始。在高蛋白质浓度下的可逆热结构转变的发现，预计将使我们能够分析在热结构转变的蛋白质的折叠和去折叠过程的多样性。
To clarify mechanisms of folding and unfolding of proteins, many studies of thermal denaturation of proteins have been carried out at low protein concentrations because in many cases thermal denaturation accompanies a great tendency of aggregation. As small-angle x-ray scattering (SAXS) measurements are liable to use low-concentration solutions of proteins to avoid aggregation, SAXS has been regarded as very difficult to observe detailed features of thermal structural transitions such as intramolecular structural changes. By using synchrotron radiation SAXS, we have found that the presence of repulsive interparticle interaction between proteins can maintain solute particles separately to prevent further aggregation in thermal denaturation processes and that under such conditions the thermal structural transition of hen egg-white lysozyme (HEWL) holds high reversibility even at 5% w/v HEWL below pH similar to 5. Because of the use of the high concentration of the solutions, the scattering data has enough high-statistical accuracy to discuss the thermal structural transition depending on the structural hierarchy. Thus, the tertiary structural change of HEWL starts from mostly the onset temperature determined by the differential scanning calorimetry measurement, which accompanies a large heat absorption, whereas the intramolecular structural change, corresponding to the interdomain correlation and polypeptide chain arrangement, starts much prior to the above main transition. The present finding of the reversible thermal structural transitions at the high protein concentration is expected to enable us to analyze multiplicity of folding and unfolding processes of proteins in thermal structural transitions.