Patra Malay, Mukhopadhyay Chaitali, Chakrabarti Abhijit
Chemistry Department, University of Calcutta, Kolkata, West Bengal, India.
Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India.
PLoS One. 2015 Jan 24;10(1):e0116991. doi: 10.1371/journal.pone.0116991. eCollection 2015.
We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔGuH20) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.
我们分别研究了两种同源膜骨架蛋白，即红细胞血影蛋白和非红细胞血影蛋白，在尿素和盐酸胍（GuHCl）存在下展开过程中其各自二聚体和四聚体形式的构象稳定性。荧光和圆二色性（CD）光谱已用于研究内在色氨酸荧光、各向异性、远紫外CD以及结合的1-苯胺基萘-8-磺酸（ANS）的外在荧光的变化。两种蛋白质的化学展开都是可逆的，并且可以描述为两态转变。折叠的红细胞血影蛋白和非红细胞血影蛋白直接转变为未折叠的单体，不形成任何中间体。荧光猝灭、各向异性、ANS结合和动态光散射数据表明，在低浓度变性剂（高达1M）存在下，氢键网络和范德华相互作用在诱导四级和三级结构变化中起作用，而亚基不会完全解离。这是关于两种大型蠕虫状多结构域蛋白遵循在小型球状蛋白中常见的双重规则的首次报道。二聚体血影蛋白的稳定自由能（ΔGuH20）比四聚体形式低20千卡/摩尔。
