Sen Debasish, Mandal Dipak K
Department of Chemistry, Presidency University, Kolkata, India.
Protein Pept Lett. 2014;21(1):80-9. doi: 10.2174/09298665113209990088.
The unfolding of dimeric Erythrina cristagalli lectin (ECL) has been investigated and compared under different denaturing conditions in presence of chemical denaturant, guanidine hydrochloride (GdnHCl) and fluoroalcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP). The GdnHCl-induced unfolding exhibits three-state mechanism involving structured intermediate that corresponds to tertiary monomer. The intermediate has been characterized by 8- anilino-1-naphthalenesulfonate (ANS) binding, which shows ~ 30 fold increase in ANS fluorescence and selective chemical modification with N-bromosuccinimide when Trp 45 and Trp 207 are possibly oxidized. The results are supported by red edge excitation shift (10 nm), acrylamide quenching and phosphorescence studies which give a (0,0) band at 412.6 nm. TFE and HFIP show differing roles and characteristics of ECL unfolding. In TFE, but not in HFIP, a molten globulelike monomeric intermediate is formed, being characterized by ANS binding and concentration dependent studies. TFEand HFIP-induced secondary structure changes of ECL, as monitored by far-UV CD, show that conversion of β-sheet to α-helix occurs at lower HFIP concentration compared to TFE perturbation, helical content reaching to 65 % in 80 % HFIP and 53 % in 90 % TFE. Temperature-dependent studies reveal that induced helix entails reduced thermal stability. FTIR results show partial β-sheet to α-helix conversion but with quantitative yield. The tryptophan environment of TFE- and HFIP-induced states is dissimilar involving oxidation of four and three tryptophans respectively, and also differs from the fully unfolded state in GdnHCl when all five tryptophans undergo oxidation. The results offer insights into the unfolding problem of ECL.
在存在化学变性剂盐酸胍（GdnHCl）、氟代醇三氟乙醇（TFE）和六氟异丙醇（HFIP）的情况下，研究并比较了二聚体刺桐凝集素（ECL）在不同变性条件下的去折叠过程。GdnHCl诱导的去折叠呈现三态机制，涉及对应于三级单体的结构化中间体。该中间体已通过8-苯胺基-1-萘磺酸盐（ANS）结合进行了表征，当色氨酸45和色氨酸207可能被氧化时，ANS荧光增加约30倍，并通过N-溴代琥珀酰亚胺进行选择性化学修饰。这些结果得到了红边激发位移（10 nm）、丙烯酰胺猝灭和磷光研究的支持，这些研究在412.6 nm处给出了一个（0,0）带。TFE和HFIP在ECL去折叠过程中表现出不同的作用和特征。在TFE中（但不在HFIP中）形成了一种类似熔球态的单体中间体，通过ANS结合和浓度依赖性研究对其进行了表征。通过远紫外圆二色性监测，TFE和HFIP诱导的ECL二级结构变化表明，与TFE扰动相比，在较低的HFIP浓度下就会发生β-折叠向α-螺旋的转变，在80%的HFIP中螺旋含量达到65%，在90%的TFE中达到53%。温度依赖性研究表明，诱导形成的螺旋导致热稳定性降低。傅里叶变换红外光谱结果显示部分β-折叠向α-螺旋的转变，但产率是定量的。TFE和HFIP诱导状态下的色氨酸环境不同，分别涉及四个和三个色氨酸的氧化，并且也不同于GdnHCl诱导的完全去折叠状态，在GdnHCl中所有五个色氨酸都会被氧化。这些结果为ECL的去折叠问题提供了见解。
