Pawar S A, Deshpande V V
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Eur J Biochem. 2000 Nov;267(21):6331-8. doi: 10.1046/j.1432-1327.2000.01686.x.
Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.
利用内源荧光、荧光猝灭、二阶导数光谱、疏水染料（1-苯胺基-8-萘磺酸盐）结合及圆二色技术，对来自链霉菌属NCIM 2730的四聚体葡萄糖/木糖异构酶（GXI）的酸诱导去折叠过程进行了研究。不同温度下GXI色氨酸荧光的pH依赖性表明，在pH 5.0和pH 3.0时存在两种稳定中间体。pH 3.2的中间体为二聚体，呈现出类熔球状态的特征，如存在类似天然的二级结构、三级结构丧失、疏水口袋暴露增加、酪氨酸残基微环境改变以及丙烯酰胺淬灭的可及性增加。对pH 5.0时GXI的荧光和圆二色研究表明，在天然态到熔球态转变过程中涉及一个部分折叠的中间体状态。与熔球态相比，部分折叠的中间体状态保留了相当多的二级和三级结构。该状态的特征在于其疏水染料结合能力，其小于熔球态，但大于天然态。该状态与熔球态共享二聚体状态，但如瑞利光散射研究所表明的，易于形成聚集体。基于这些结果，GXI的去折叠途径可表示为：N→PFI→MG→U；其中N是pH 7.5时的天然态；PFI是pH 5.0时的部分折叠中间体状态；MG是pH 3.2时的熔球态，U是在6 M盐酸胍存在下获得的GXI的单体去折叠态。我们的结果证明了在多聚体α/β桶状蛋白的去折叠途径上存在部分折叠态和熔球态。
