TWO-DIMENSIONAL NMR AND PHOTO-CIDNP STUDIES OF THE INSULIN MONOMER - ASSIGNMENT OF AROMATIC RESONANCES WITH APPLICATION TO PROTEIN FOLDING, STRUCTURE, AND DYNAMICS
TWO-DIMENSIONAL NMR AND PHOTO-CIDNP STUDIES OF THE INSULIN MONOMER - ASSIGNMENT OF AROMATIC RESONANCES WITH APPLICATION TO PROTEIN FOLDING, STRUCTURE, AND DYNAMICS
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DOI：
10.1021/bi00451a046
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发表时间：
1989-12-12
期刊：
影响因子：
2.9
通讯作者：
NEURINGER, LJ
中科院分区：
文献类型：
--
作者：
WEISS, MA;NGUYEN, DT;NEURINGER, LJ
通过对化学修饰和基因改变变体（包括与糖尿病相关的突变胰岛素 (PheB25.fwdarw.Leu)）的比较研究，将胰岛素单体的芳香族 1H NMR 共振指定为 500 MHz。观察到两个组氨酸、三个苯丙氨酸和四个酪氨酸存在于不同的局部环境中；他们的任务为三级结构、蛋白质动力学和蛋白质折叠的研究提供了敏感的标记。还通过光化学诱导动态核极化（photo-CIDNP）研究了酪氨酸残基的环境，并分析了胰岛素晶体结构中堆积约束的关系。随着蛋白质浓度的增加，观察到涉及特定 B 链相互作用的二聚化，并且表明二聚化取决于温度、pH 和溶剂组成。在单体中，酰胺共振线宽观察到较大变化，表明构象亚态之间存在中间交换；这些亚状态可能与在不同晶态中观察到的构象变化有关，并推测发生在激素-受体复合物中。对残基 B29 和 A1（微型胰岛素原）之间含有肽键的胰岛素类似物的比较研究提供了溶液中多种构象的额外证据。该类似物在天然胰岛素为单体的条件下形成二聚体和高阶寡聚体，这表明 B29-A1 肽键稳定了有利于二聚化的构象底物。在天然胰岛素原的相应研究中没有观察到这种稳定性，其中 35 个残基的连接肽连接残基 B30 和 A1；这种延伸的系链可能太柔韧而无法限制 B 链的构象。胰岛素原和微型胰岛素原之间的差异提示了观察到的结构机制，即完全还原的 B29-A1 类似物比胰岛素原更有效地折叠以形成正确的二硫键模式。这些结果根据现有的晶体结构与胰岛素的分子力学计算相关进行讨论。
The aromatic 1H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25.fwdarw.Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constraints in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. In the monomer large variations are observed in the line widths of amide resonances, suggesting intermediate exchange among conformational substates; such substates may relate to conformational changes observed in different crystal states and proposed to occur in the hormone-receptor complex. Additional evidence for multiple conformations in solution is provided by comparative studies of an insulin analogue containing a peptide bond between residues B29 and A1 (mini-proinsulin). This analogue forms dimers and higher-order oligomers under conditions in which native insulin is monomeric, suggesting that the B29-A1 peptide bond stabilizes a conformational substate favorable for dimerization. Such stabilization is not observed in corresponding studies of native proinsulin, in which a 35-residue connecting peptide joins residues B30 and A1; this extended tether is presumably too flexible to constrain the conformation of the B-chain. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures.