Siebendritt R, Sharma A K, Rudolph R, Jaenicke R
Institut für Biophysik und Physikalische Biochemie, Universitä Regensburg.
Biol Chem Hoppe Seyler. 1991 Jan;372(1):23-6. doi: 10.1515/bchm3.1991.372.1.23.
Fast protein liquid chromatography was effectively applied to analyse the folding mechanism of gamma-II-crystallin from calf eye-lens. The protein undergoes a bimodal folding/unfolding transition, according to a three-state model: N in equilibrium I in equilibrium D where N, I, and D stand for the native, intermediate and denatured states (R. Rudolph, R. Siebendritt, G. Nesslauer, A.K. Sharma & R. Jaenicke (1990) Proc. Natl. Acad. Sci. USA 87, 4625-4629). Using Superose 12 HR 10/30, the intermediate with the N-terminal domain intact, and the C-terminal domain unfolded, could be separated from the native protein. The N----I transition is sufficiently slow to allow kinetic measurements, following the variation of the respective peak-heights during denaturation/renaturation. The corresponding relaxation times are in agreement with kinetic data based on the change in fluorescence emission accompanying the N in equilibrium I transition.
快速蛋白质液相色谱法被有效地应用于分析来自小牛眼晶状体的γ-II-晶状体蛋白的折叠机制。根据三态模型，该蛋白质经历双峰折叠/去折叠转变：N处于平衡态I处于平衡态D，其中N、I和D分别代表天然态、中间态和变性态（R. 鲁道夫、R. 西本德里特、G. 内斯劳尔、A.K. 夏尔马和R. 耶尼克（1990年）《美国国家科学院院刊》87, 4625 - 4629）。使用Superose 12 HR 10/30，可以将N端结构域完整而C端结构域展开的中间态与天然蛋白质分离。N→I转变足够缓慢，从而能够在变性/复性过程中跟踪各个峰高的变化来进行动力学测量。相应的弛豫时间与基于N处于平衡态I转变时伴随的荧光发射变化的动力学数据一致。
