Nakagawa Kanako, Yamada Yoshiteru, Fujiwara Kazuo, Ikeguchi Masamichi
Department of Bioinformatics, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
J Mol Biol. 2007 Apr 6;367(4):1205-14. doi: 10.1016/j.jmb.2007.01.053. Epub 2007 Jan 25.
Equine beta-lactoglobulin forms a compact intermediate at an acidic pH (A state). It also forms an expanded and helical conformation at low temperatures (C state). The structure of a single disulfide mutant C66A/C160A is similar to the A state in the presence of salts, while it is similar to the C state at low anion concentrations. We have investigated the temperature-dependent change in the secondary structure using circular dichroism and proline scanning mutagenesis. At low anion concentrations, the helical content increased linearly as temperature decreased. In the presence of salts, the A state was cooperatively transformed into the C state at low temperatures. This suggests the importance of hydrophobic interactions for stabilizing the A state. Peptides encompassing native-like and non-native alpha-helices were synthesized to investigate the interactions responsible for helix formation in the A and C states. These did not form stable helices, indicating that not only the helices in the A state but also the helices in the C state are stabilized by long-range interactions. A longer fragment, CHIBL, which encompasses the structured region in the A and C states, showed a helical structure. Proline-substituted mutants of CHIBL showed CD spectral changes similar to the corresponding mutants of the full-length protein in the C state. Therefore, CHIBL has a structure similar to the corresponding region of the full-length protein in the C state. This result indicates that interactions responsible for helix formation in the C state reside in the sequence of CHIBL, and that the sequences outside CHIBL are essential for secondary structure formation in the A state.
马β-乳球蛋白在酸性pH值下形成一种紧密的中间体（A状态）。它在低温下也会形成一种伸展的螺旋构象（C状态）。单个二硫键突变体C66A/C160A在有盐存在时其结构类似于A状态，而在低阴离子浓度下则类似于C状态。我们使用圆二色性和脯氨酸扫描诱变研究了二级结构随温度的变化。在低阴离子浓度下，随着温度降低螺旋含量呈线性增加。在有盐存在时，A状态在低温下协同转变为C状态。这表明疏水相互作用对于稳定A状态很重要。合成了包含天然样和非天然α-螺旋的肽段，以研究在A和C状态下负责螺旋形成的相互作用。这些肽段没有形成稳定的螺旋，这表明不仅A状态下的螺旋，而且C状态下的螺旋都是通过长程相互作用稳定的。一个更长的片段CHIBL，它包含A和C状态下的结构化区域，呈现出螺旋结构。CHIBL的脯氨酸取代突变体显示出与C状态下全长蛋白相应突变体相似的圆二色光谱变化。因此，CHIBL具有与C状态下全长蛋白相应区域相似的结构。这一结果表明，在C状态下负责螺旋形成的相互作用存在于CHIBL序列中，并且CHIBL以外的序列对于A状态下的二级结构形成至关重要。
