Zerovnik E, Virden R, Jerala R, Turk V, Waltho J P
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Proteins. 1998 Aug 15;32(3):296-303.
The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60+/-5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved "burst" phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6s) detected by all the spectroscopic probes, which occurred subsequent to an initial "burst" phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed.
已经通过几种光谱探针研究了人丝氨酸蛋白酶抑制剂B的折叠过程。比较了通过近紫外和远紫外圆二色性、酪氨酸荧光以及外在探针ANS荧光获得的停流曲线。远紫外圆二色性（CD）中大部分（60±5%）的天然信号在10毫秒内出现在一个未解析的“爆发”阶段，随后是一个快速阶段（t = 83毫秒）和一个缓慢阶段（t = 25秒），其幅度分别为30%和10%。类似的快速和缓慢阶段在近紫外CD、ANS荧光和酪氨酸荧光中也很明显。相比之下，结构非常相似的人丝氨酸蛋白酶抑制剂A，通过所有光谱探针检测到其折叠过程只有一个动力学阶段（t = 6秒），该阶段发生在远紫外CD观察到的初始“爆发”阶段之后。有趣的是，尽管这两种同源物结构相似，但它们的折叠方式不同，而且稳定性较差的人丝氨酸蛋白酶抑制剂B折叠速度快一个数量级（比较非脯氨酸限制阶段）。为了获得更多关于丝氨酸蛋白酶抑制剂B折叠机制的信息，通过几种光谱探针研究了pH值和三氟乙醇（TFE）对快速和缓慢阶段的影响。如果在7%的TFE存在下进行折叠，则会观察到速率加速和机制差异。
