Zerovnik E, Jerala R, Virden R, Kroon Zitko L, Turk V, Waltho J P
Department of Biochemistry and Molecular Biology, Josef Stefan Institute, Ljubljana, Slovenia.
Proteins. 1998 Aug 15;32(3):304-13. doi: 10.1002/(sici)1097-0134(19980815)32:3<304::aid-prot6>3.0.co;2-h.
It has been shown that human stefin B exhibits molten globule intermediates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Zerovnik et al., Proteins 32:296-303), the kinetics measured by circular dichroism (CD) and fluorescence were correlated. In the present work the kinetics of folding were monitored by tyrosine fluorescence, ANS fluorescence, and, for certain reactions, far ultraviolet (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid denatured state at pH 1.8+/-0.2, an acid molten globule intermediate I1 (pH 3.3+/-0.1, low salt), a more structured acid molten globule intermediate I2 (pH 3.3+/-0.1, 0.42 M NaCl), and the TFE state (pH 3.3+/-0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identical. This does not apply to the second acid molten globule intermediate I2, which demonstrates a higher rate of folding by a factor of 270. Different energy of activation and pH dependence were found for folding from states I1 or I2.
研究表明，人源丝氨酸蛋白酶抑制剂B在被酸或盐酸胍（GuHCl）变性时会呈现熔球态中间体。在三氟乙醇（TFE）存在的情况下，它会转变为高度螺旋的状态。在我们关于其折叠机制的第一项研究中（泽罗夫尼克等人，《蛋白质》32:296 - 303），通过圆二色性（CD）和荧光测量的动力学是相关的。在本研究中，通过酪氨酸荧光、ANS荧光以及对于某些反应通过远紫外（UV）CD来监测折叠动力学。折叠从3.45 M GuHCl中的未折叠状态、pH 1.8±0.2的酸变性状态、酸熔球态中间体I1（pH 3.3±0.1，低盐）、结构更紧密的酸熔球态中间体I2（pH 3.3±0.1，0.42 M NaCl）以及TFE状态（pH 3.3±0.1，42% TFE）开始。已经发现，所有变性状态，包括GuHCl、TFE、酸变性状态和酸熔球态中间体I1，只要最终条件相同，就以相同的动力学进行折叠。但这不适用于第二个酸熔球态中间体I2，它的折叠速率要高270倍。从状态I1或I2折叠时发现了不同的活化能和pH依赖性。
