Fatima Sadaf, Ahmad Basir, Khan Rizwan Hasan
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
Arch Biochem Biophys. 2006 Oct 15;454(2):170-80. doi: 10.1016/j.abb.2006.08.014. Epub 2006 Aug 31.
Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.
伴刀豆球蛋白A（Con A）在pH 5时以二聚体状态存在。在20 - 60%（v/v）的2,2,2 - 三氟乙醇（TFE）和2 - 40%（v/v）的1,1,1,3,3,3 - 六氟异丙醇（HFIP）浓度范围内，pH 5.0的Con A会出现明显聚集。然而，当使用琥珀酰化Con A时，在整个氟代醇浓度范围（0 - 90% v/v TFE和HFIP）内均未观察到聚集现象，并且形成了稳定的α - 螺旋结构。研究还发现，琥珀酰化形式的Con A能防止/减少温度诱导的浓度依赖性聚集。文中讨论了残基间静电排斥在防止疏水驱动聚集方面可能发挥的作用。结果表明，蛋白质的琥珀酰化导致其在抵抗酒精诱导和温度诱导的浓度依赖性聚集方面具有更高的稳定性（β - 折叠和α - 螺旋形式均如此），这一观察结果可能在形成淀粉样蛋白的蛋白质中发挥重要作用。通过圆二色性（CD）、荧光发射光谱、疏水染料ANS（8 - 苯胺基萘 - 1 - 磺酸）结合等方法研究了TFE和HFIP对二聚体蛋白琥珀酰化Con A构象的影响。远紫外CD作为二级结构的探针显示，在低浓度的两种醇（10% v/v TFE和4% v/v HFIP）存在下，天然二级结构会丧失。加入更高浓度的这些醇后，琥珀酰化Con A表现出从β - 折叠结构向α - 螺旋结构的转变。色氨酸内源荧光研究、ANS结合以及近紫外CD实验表明，在60%（v/v）TFE和30%（v/v）HFIP浓度下，该蛋白更加伸展，具有更多暴露的疏水表面且三级结构高度破坏。综上所述，这些结果可能表明，TFE和HFIP在琥珀酰化Con A中低浓度和高浓度时会诱导两种中间状态。
