Department of Chemistry, Presidency University, 86/1 College Street, Kolkata 700 073, India.
Department of Chemistry, Presidency University, 86/1 College Street, Kolkata 700 073, India.
Biochimie. 2014 Apr;99:138-45. doi: 10.1016/j.biochi.2013.11.022. Epub 2013 Dec 4.
The sequence of unfolding events of dimeric banana lectin (Banlec), as induced by guanidine hydrochloride (GdnHCl), has been investigated by size-exclusion HPLC, fluorescence, far-UV CD, low temperature phosphorescence and selective chemical modification. 8-Anilino-1-naphthalenesulfonate (ANS) binding indicates a structured unfolding intermediate which has been characterized as dissociated monomer by size-exclusion chromatography. Interestingly, the unfolding elution pattern reveals two distinct unfolded states. One is a usual random coil. The other represents a novel species having elution behavior and structural compactness (Stokes radius) similar to dissociated monomer but showing no regular secondary structure as determined by far-UV CD, thus resembling a natively unfolded state. N-Bromosuccinimide (NBS) oxidation shows that single tryptophan residue remains unmodified in dissociated monomer intermediate while the same is oxidized in natively unfolded-like species. Such difference in tryptophan environment in these species is supported by acrylamide quenching studies, and phosphorescence results at 77 K which show a blue-shift of (0,0) band from 414.8 nm to 409.2 nm. The present results reveal subtlety of structural characteristics of unfolded states of Banlec in GdnHCl, which provide important insight in protein unfolding reaction.
二聚体香蕉凝集素（Banlec）在盐酸胍（GdnHCl）诱导下的展开事件序列已通过尺寸排阻 HPLC、荧光、远紫外 CD、低温磷光和选择性化学修饰进行了研究。8-苯胺-1-萘磺酸（ANS）结合表明存在结构展开中间态，通过尺寸排阻色谱将其鉴定为解离单体。有趣的是，展开洗脱模式揭示了两种不同的展开状态。一种是通常的无规卷曲。另一种代表一种新型物种，其洗脱行为和结构紧凑性（Stokes 半径）类似于解离单体，但远紫外 CD 确定没有规则的二级结构，因此类似于天然无折叠状态。N-溴代丁二酰亚胺（NBS）氧化表明，单个色氨酸残基在解离单体中间态保持未修饰，而在天然无折叠样物种中则被氧化。这些物种中色氨酸环境的差异得到丙烯酰胺猝灭研究和 77 K 磷光结果的支持，磷光结果显示（0,0）带从 414.8 nm 蓝移至 409.2 nm。目前的结果揭示了 GdnHCl 中 Banlec 无折叠态结构特征的细微差别，为蛋白质展开反应提供了重要的见解。
