ENGINEERING STABILITY OF THE INSULIN MONOMER FOLD WITH APPLICATION TO STRUCTURE-ACTIVITY-RELATIONSHIPS
ENGINEERING STABILITY OF THE INSULIN MONOMER FOLD WITH APPLICATION TO STRUCTURE-ACTIVITY-RELATIONSHIPS
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DOI：
10.1021/bi00091a031
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发表时间：
1993-10-12
期刊：
影响因子：
2.9
通讯作者：
HAVELUND, S
中科院分区：
文献类型：
--
作者：
KAARSHOLM, NC;NORRIS, K;HAVELUND, S
为了评估胰岛素分子选定区域的生物活性和结构稳定性之间可能的关系，我们结合近紫外和远紫外圆二色性（CD）分析了盐酸胍诱导的一系列突变胰岛素的可逆展开。基于二态变性方案合理地描述了展开曲线；然而，观察近紫外和远紫外 CD 检测到的展开之间的细微差异表明可能存在中间体。详细分析了胰岛素分子的三个区域对折叠稳定性的贡献，即中央 B 链螺旋、NH2 末端 A 链螺旋和 B25-B30 延长链区域。通过中央 B 链螺旋 N 帽和 NH2 末端 A 链螺旋 C 帽的突变，可显着增强折叠稳定性。在这些区域中赋予稳定性增加的突变与导致生物活性增强的突变相同。相比之下，对于分子 B25-B30 区域修饰的胰岛素种类，我们观察到整体折叠稳定性和生物活性之间没有相关性。发现所检查的三个区域中的突变以几乎独立的方式影响稳定性，并且通常发现稳定突变可以增强展开转变的协同性。我们得出的结论是，高效胰岛素（即 HisA8、ArgA8、GluB10 和 AspB10）会引起活性增强，因为这些突变稳定了对受体识别至关重要的结构基序。
To evaluate the possible relationship between biological activity and structural stability in selected regions of the insulin molecule, we have analyzed the guanidine hydrochloride induced reversible unfolding of a series of mutant insulins using a combination of near- and far-UV circular dichroism (CD). The unfolding curves are reasonably described on the basis of a two-state denaturation scheme; however, the observation of subtle differences between near- and far-UV CD detected unfolding indicates that intermediates may be present. Three regions of the insulin molecule are analyzed in detail with respect to their contribution to folding stability, i.e., the central B-chain helix, the NH2-terminal A-chain helix, and the B25-B30 extended chain region. Considerable enhancement of folding stability is engineered by mutations at the N-cap of the central B-chain helix and at the C-cap of the NH2-terminal A-chain helix. Mutations that confer increased stability in these regions are identical to those that lead to enhanced biological activity. In contrast, for insulin species modified in the B25-B30 region of the molecule, we observe no correlation between global folding stability and bioactivity. Mutations in the three regions examined are found to affect stability in a nearly independent fashion, and stabilizing mutations are generally found to enhance the cooperativity of the unfolding transition. We conclude that highly potent insulins (i.e., HisA8, ArgA8, GluB10, and AspB10) elicit enhanced activity because these mutations stabilize structural motifs of critical importance for receptor recognition.