Temperature-jump induced fast refolding of cold-unfolded protein.
Temperature-jump induced fast refolding of cold-unfolded protein.
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温度突变诱导冷解折叠蛋白质的快速重折叠。
DOI：
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发表时间：
1996
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影响因子：
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通讯作者：
B. Nölting
中科院分区：
文献类型：
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作者：
B. Nölting
只有微秒存活时间的瞬时蛋白质结构可以通过冷变性蛋白质的温度跳跃在单个残基的分辨下得到解决[Nölting，B.，Golbik，R.，and Fersht，A.R.(1995)Proc.娜塔莉。阿卡德。SCI。美国92,10668-10672]。这里展示了如何将这种方法扩展到位于主折叠过渡态和自然态之间的反应坐标上的中间体和过渡态的研究。当牛β-乳球蛋白A在有利于冷展开和部分冷展开的条件下通过温度从-4℃到2℃的跳跃快速复性时，观察到快速折叠动力学，其速率常数约为10 0 S-1。在T跃迁前的低温孕育时间下，弛豫幅度呈单指数函数衰减。衰变的速率常数与主要展开转变的速率常数相匹配，表明在冷变性反应的早期，β-乳球蛋白被动力学地捕获在部分未折叠的中间状态。尽管只有很小的荧光变化，但在温度跃升后约10ms内进行重折叠时，需要相当程度的溶剂排斥和疏水表面的掩埋，这表明分子收缩。
Transient protein structures with only microsecond live times may be solved at the resolution of single residues by temperature jumping of cold denatured protein [Nölting, B., Golbik, R., and Fersht, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 10668-10672]. Here it is shown how this method may be extended to the study of intermediates and transition states which are located on the reaction coordinate between the main folding transition state and the native state. When incubating bovine beta-lactoglobulin A under conditions which favour cold unfolding and rapidly refolding the partially cold unfolded protein by a temperature jump from -4 to 2 degrees C, a fast folding kinetics with a rate constant of about 100 s-1 is observed. The amplitude of the relaxation decays following a single exponential function of the time of incubation at low temperature before the T-jump. The rate constant of decay matches the rate constant of the main unfolding transition, showing that early in the cold denaturation reaction, beta-lactoglobulin is kinetically trapped in a partially unfolded intermediate state. Despite the only small fluorescence change, refolding within about 10 ms after a temperature jump involves a considerable degree of solvent exclusion and burial of hydrophobic surface, suggesting a contraction of the molecule.