Khurana R, Udgaonkar J B
National Centre for Biological Sciences, Indian Institute of Science Campus, Bangalore.
Biochemistry. 1994 Jan 11;33(1):106-15. doi: 10.1021/bi00167a014.
The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.
芽孢杆菌RNA酶抑制剂（barstar）是解淀粉芽孢杆菌中RNA酶（barnase）的细胞内抑制剂，其折叠过程已通过平衡去折叠方法进行了研究。结果表明，barstar存在两种构象：A构象，存在于pH值低于4的环境中；N状态，存在于pH值高于5的环境中。A构象和N状态之间的转变是完全可逆的。利用紫外吸收光谱、荧光光谱和圆二色光谱对这两种构象进行了研究。在220nm处测得的A构象的平均残基椭圆率是N状态的60%，并且A构象具有一些熔融球状构象所预期的性质。使用1-苯胺基-8-萘磺酸盐的荧光能量转移实验表明，A构象中的三个色氨酸残基中至少有一个可与水接触。令人惊讶的是，需要高浓度的变性剂才能使A构象去折叠。对于盐酸胍变性，通过圆二色性测量的A构象在pH 3时协同去折叠转变的中点为3.7±0.1M，这明显高于在pH 7时N状态观察到的2.0±0.1M的值。盐酸胍或尿素使A构象去折叠的过程很复杂，不能令人满意地拟合为两态（A<==>U）去折叠模型。荧光监测的三级结构在圆二色性监测的二级结构之前发生熔融，并且在A构象的去折叠途径中必定存在一个平衡去折叠中间体。
