Yamada Yoshiteru, Yajima Takeo, Fujiwara Kazuo, Arai Munehito, Ito Kazuki, Shimizu Akio, Kihara Hiroshi, Kuwajima Kunihiro, Amemiya Yoshiyuki, Ikeguchi Masamichi
Department of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
J Mol Biol. 2005 Jul 8;350(2):338-48. doi: 10.1016/j.jmb.2005.05.003.
The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.
通过圆二色性（CD）在-15℃至85℃的温度范围内研究了马β-乳球蛋白（ELG）的热展开转变。在2M尿素存在下，随着温度升高和降低均观察到协同展开转变。CD光谱表明，ELG的热变性和冷变性状态具有大量二级结构，但缺乏侧链的持久三级堆积。为了阐明热变性或冷变性状态与先前表征的酸变性（A）状态之间的关系，我们试图观察pH 1.5时CD光谱的温度依赖性。热变性状态下的CD光谱与A状态下的相似。A状态下的CD光谱不会随着温度升高而协同变化。这些结果表明，热变性状态和A状态是相同的结构状态。另一方面，酸性pH下的CD强度随着温度降低而协同增加。低温和酸性pH下的CD光谱与冷变性状态下的一致。因此，冷变性状态与热变性状态或A状态不同，并且ELG在冷变性状态下具有大量非天然α-螺旋。小角X射线散射和分析超速离心表明，与A状态下的紧密球状构象相比，ELG在冷变性状态下呈现出扩展的链状构象。讨论了部分折叠状态下分子大小与螺旋含量之间的关系。
