Qin Z, Hu D, Shimada L, Nakagawa T, Arai M, Zhou J M, Kihara H
Department of Physics, Kansai Medical University, Hirakata, Japan.
FEBS Lett. 2001 Nov 2;507(3):299-302. doi: 10.1016/s0014-5793(01)02886-1.
Refolding of bovine beta-lactoglobulin was studied by stopped-flow circular dichroism at subzero temperatures. In ethylene glycol 45%-buffer 55% at -15 degrees C, the isomerization rate from the kinetic intermediate rich in alpha-helix to the native state is approximately 300-fold slower than that at 4 degrees C in the absence of ethylene glycol, whereas the initial folding is completed within the dead time of the stopped-flow apparatus (10 ms). At -28 degrees C, we observed at least three phases; the fastest process, accompanied by an increase of alpha-helix content, is completed within the dead time of the stopped-flow apparatus (10 ms), the second phase, accompanied by an increase of alpha-helix content with the rate of 2 s(-1), and the third phase, accompanied by a decrease of alpha-helix content. This last phase, corresponding to the isomerization process at -15 degrees C described above, was so slow that we could not monitor any changes within 4 h. Based on the findings above, we propose that rapid alpha-helix formation and their concurrent collapse are common even in proteins rich in beta-structure in their native forms.
通过在零下温度下的停流圆二色性研究了牛β-乳球蛋白的复性。在-15℃的45%乙二醇-55%缓冲液中，从富含α-螺旋的动力学中间体向天然态的异构化速率比在4℃且不存在乙二醇时慢约300倍，而初始折叠在停流装置的死时间（10毫秒）内完成。在-28℃时，我们观察到至少三个阶段；最快的过程伴随着α-螺旋含量的增加，在停流装置的死时间（10毫秒）内完成，第二阶段伴随着α-螺旋含量以2 s⁻¹的速率增加，第三阶段伴随着α-螺旋含量的减少。最后这个阶段，对应于上述-15℃的异构化过程，非常缓慢，以至于我们在4小时内无法监测到任何变化。基于上述发现，我们提出即使在天然形式富含β-结构的蛋白质中，快速的α-螺旋形成及其同时的折叠也是常见的。
