Maintenance of the B-chain beta-turn in [GlyB24] insulin mutants: a steady-state fluorescence anisotropy study.
Maintenance of the B-chain beta-turn in [GlyB24] insulin mutants: a steady-state fluorescence anisotropy study.
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DOI：
10.1021/bi9624144
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发表时间：
1997-03
期刊：
影响因子：
2.9
通讯作者：
I. Pittman;S. Nakagawa;H. Tager;D. Steiner
中科院分区：
文献类型：
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作者：
I. Pittman;S. Nakagawa;H. Tager;D. Steiner
[GlyB 24]胰岛素是一种新的胰岛素类似物，其保持几乎完全的生物活性[Mirmira，R. G.，& Tager，H. S.（1989）J.Biol.Chem.264，6349-6354]，尽管其结构（如通过2D NMR测定）显示特征性B链β-转角的完全丧失[Hua，Q. X.，Shoelson，S. E、Kochoyan，M.，&韦斯，M. A.（1991）Nature 354，238-241]，其在天然胰岛素中允许延伸的B链C末端区域相对于中心B链螺旋折叠。在这些研究中，对Dahan取代的类似物[TrpB 25]胰岛素和[GlyB 24，TrpB 25]胰岛素已用于研究B链C末端区域的结构，并证明[GlyB 24]胰岛素突变体维持正常B链构象的程度与天然（PheB 24）相当在具有[GlyB 24，TrpB 25-α-羧酰胺]的溶液中，如通过这些方法评估的，TrpB 25-α-甲酰胺]DPI比其相应的PheB 24-类似物（[TrpB 25-α-甲酰胺]DPI）更紧密地折叠。各向异性测量的结果与[GlyB 24]胰岛素的高亲和力受体结合与生理条件下B链β转角的部分维持之间存在相关性一致。因此，我们得出结论，只有具有或能够容易地呈现这种定向结构的类似物才能与胰岛素受体形成高亲和力结合复合物。
[GlyB24]insulin is a novel insulin analog which maintains nearly full biological activity [Mirmira, R. G., & Tager, H. S. (1989) J. Biol. Chem. 264, 6349-6354] even though its structure, as determined by 2D NMR, shows complete loss of the characteristic B-chain beta-turn [Hua, Q. X., Shoelson, S. E., Kochoyan, M., & Weiss, M. A. (1991) Nature 354, 238-241], which in native insulin allows the extended B-chain C-terminal region to fold against the central B-chain helix. In these studies, steady-state anisotropy measurements and fluorescence quenching analysis of the tryptophan-substituted analogs [TrpB25]insulin and [GlyB24,TrpB25]insulin have been used to study the structure of the C-terminal region of the B-chain and have demonstrated that [GlyB24]insulin mutants maintain the normal B-chain conformation to a degree comparable to that of native (PheB24) insulin at neutral pH. The tryptophan-substituted, B-chain C-terminally truncated analogs [TrpB25-alpha-carboxamide]despentapeptide(B26-B30)-insulin (DPI) and [GlyB24,TrpB25-alpha-carboxamide]DPI also significantly retain the characteristic insulin B-chain fold in solution with [GlyB24,TrpB25-alpha-carboxamide]DPI being more tightly folded than its corresponding PheB24-analog ([TrpB25-alpha-carboxamide]DPI), as assessed by these methods. The results of anisotropy measurements are consistent with the existence of a correlation between the high-affinity receptor binding of [GlyB24]insulin and the partial maintenance of the B-chain beta-turn under physiologic conditions. Thus we conclude that only analogs which possess, or can readily assume, this oriented structure can form high-affinity binding complexes with insulin receptor.