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首页> 外文期刊>Journal of Biochemistry >Different Molten Globule-like Folding Intermediates of Hen Egg White Lysozyme Induced by High pH and Tertiary Butanol
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Different Molten Globule-like Folding Intermediates of Hen Egg White Lysozyme Induced by High pH and Tertiary Butanol

机译：高pH和叔丁醇诱导的鸡卵清溶菌酶的不同熔融球状折叠中间体

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摘要

We have provided evidence that hen egg white lysozyme (HEWL) existed in α helical and β structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (～5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly β sheets. On further increasing the t-butanol concentration α helix was found to reform. We have proposed that formation of both α helical and β sheet dominated intermediate may be possible in the folding pathway of α + β protein.
机译：我们提供的证据表明，在高pH和叔丁醇存在下，鸡蛋清溶菌酶（HEWL）分别以α螺旋和β结构为主的熔融小球（MG）状态存在。圆二色性（CD），固有荧光，ANS结合和丙烯酰胺诱导的荧光猝灭技术已用于研究碱诱导的HEWL的展开以及叔丁醇对碱诱导状态的影响。在pH 12.75时，HEWL以熔融小球的形式存在，像中间体。观察到的MG样中间体的特征在于（i）保留77％的天然二级结构；（ii）与天然和完全展开状态相比，ANS的结合增强（〜5倍）；（iii）三级结构丧失如三级结构探针（近紫外光，CD和内在荧光）所示，以及（iv）丙烯酰胺淬灭研究表明，MG状态具有介于天然状态和完全未折叠状态之间的紧密度。此外，还描述了蛋白质在等电点（pI）和变性状态的结构特性。我们还表明，在45％的叔丁醇（叔丁醇）存在下，pH 7.0和11.0的HEWL（pI 11.0）呈螺旋结构存在，而对叔结构的影响不大。有趣的是，HEWL在pH 12.7时的MG状态在20％叔丁醇（v / v）下转变为另一个MG状态（MG2），其中二级结构主要是β片。随着叔丁醇浓度的进一步增加，发现α螺旋发生了改变。我们已经提出，在α+β蛋白质的折叠途径中可能同时形成α螺旋和β折叠占主导的中间体。

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