Maldonado S, Jiménez M A, Langdon G M, Sancho J
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain.
Biochemistry. 1998 Jul 28;37(30):10589-96. doi: 10.1021/bi980368x.
We have destabilized apoflavodoxin by site-specific excision of its C-terminal helix. The resulting flavodoxin fragment (Fld1-149) is compact and monomeric at pH 7.0, with spectroscopic properties of a molten globule and a low conformational stability. To study if Fld1-149 is cooperatively stabilized, we have measured the equilibrium urea unfolding by fluorescence, circular dichroism, and size-exclusion chromatography. The three techniques produced coincident unfolding curves. Furthermore, the thermal unfolding seems also to be cooperative as the same temperature of half-denaturation is obtained using fluorescence and circular dichroism. Fld1-149 displays cold denaturation. The equilibrium properties of Fld1-149 demonstrate that molten globules lacking well-defined tertiary interactions can still be cooperatively stabilized and that cooperatively may appear in protein conformations of very low stability. This suggests that protein folding intermediates, can, in principle, be cooperatively stabilized.
我们通过定点切除其C端螺旋使脱辅基黄素氧还蛋白失稳。所得的黄素氧还蛋白片段（Fld1-149）在pH 7.0时呈紧密单体状态，具有熔球态的光谱特性和较低的构象稳定性。为研究Fld1-149是否能协同稳定，我们通过荧光、圆二色光谱和尺寸排阻色谱法测量了其在尿素中的平衡去折叠过程。这三种技术得到了一致的去折叠曲线。此外，热去折叠似乎也是协同的，因为使用荧光和圆二色光谱法得到了相同的半变性温度。Fld1-149表现出冷变性。Fld1-149的平衡性质表明，缺乏明确三级相互作用的熔球态仍可协同稳定，且协同作用可能出现在稳定性极低的蛋白质构象中。这表明，蛋白质折叠中间体原则上可以协同稳定。
