Haque Md Anzarul, Ubaid-Ullah Shah, Zaidi Sobia, Hassan Md Imtaiyaz, Islam Asimul, Batra Janendra K, Ahmad Faizan
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India; Immunochemistry Lab, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
Int J Biol Macromol. 2015 Jan;72:1406-18. doi: 10.1016/j.ijbiomac.2014.10.053. Epub 2014 Nov 4.
To understand the role of five extra N-terminal residues, we prepared wild type (WT) yeast iso-1-cytochrome c (y-cyt-c) and its deletants by subsequently deleting these residues. Denaturation of all these proteins induced by LiCl was followed by observing changes in molar absorption coefficient at 405 nm (Δɛ405), the mean residue ellipticity at 222 nm ([θ]222), and the difference mean residue ellipticity at 409 nm (Δ[θ]409) near physiological pH and temperature (pH 6.0 and 25 °C). It was observed that in each case LiCl induces biphasic transition, N (native) state ↔ X (intermediate) state ↔ D (denatured) state. The intermediate (X) was characterized by the far-UV, near-UV and Soret circular dichroism, ANS (8-anilino-1-naphthalenesulfonic acid) binding and dynamic light scattering measurements. These measurements led us to conclude that X state of each protein has structural characteristics of PMG (pre-molten globule) state. Thermodynamic stability of all proteins was also determined. It was observed that the N-terminal extension stabilizes the native WT protein but it has no effect on the stability of PMG state. Another state was observed for each protein, in the presence of 0.33 M Na2SO4 at pH 2.1, which when characterized showed all structural characteristics of MG (molten globule) state.
为了了解额外的五个N端残基的作用，我们制备了野生型（WT）酵母同工酶-1-细胞色素c（y-cyt-c）及其缺失突变体，方法是依次删除这些残基。通过观察在生理pH和温度（pH 6.0和25°C）下405nm处的摩尔吸收系数变化（Δɛ405）、222nm处的平均残基椭圆率（[θ]222）以及409nm处的差异平均残基椭圆率（Δ[θ]409），跟踪LiCl诱导的所有这些蛋白质的变性过程。观察到在每种情况下，LiCl都会诱导双相转变，即N（天然）态↔X（中间）态↔D（变性）态。通过远紫外、近紫外和Soret圆二色性、ANS（8-苯胺基-1-萘磺酸）结合和动态光散射测量对中间态（X）进行了表征。这些测量结果使我们得出结论，每种蛋白质的X态具有预熔球（PMG）态的结构特征。还测定了所有蛋白质的热力学稳定性。观察到N端延伸稳定了天然WT蛋白，但对PMG态的稳定性没有影响。在pH 2.1、存在0.33 M Na2SO4的条件下，观察到每种蛋白质的另一种状态，对其进行表征时显示出熔球（MG）态的所有结构特征。
