Nielsen L, Frokjaer S, Brange J, Uversky V N, Fink A L
University of California-Santa Cruz, Department of Chemistry and Biochemistry, Santa Cruz, CA 95064, USA.
Biochemistry. 2001 Jul 27;40(28):8397-409. doi: 10.1021/bi0105983.
The molecular basis of insulin fibril formation was investigated by studying the structural properties and kinetics of fibril formation of 20 different human insulin mutants at both low pH (conditions favoring monomer/dimer) and at pH 7.4 (conditions favoring tetramer/hexamer). Small-angle X-ray scattering showed insulin to be monomeric in 20% acetic acid, 0.1 M NaCl, pH 2. The secondary structure of the mutants was assessed using far-UV circular dichroism, and the tertiary structure was determined using near-UV circular dichroism, quenching of intrinsic fluorescence by acrylamide and interactions with the hydrophobic probe 1-anilino-8-naphthalene-sulfonic acid (ANS). The kinetics of fibril formation were monitored with the fluorescent dye, Thioflavin T. The results indicate that the monomer is the state from which fibrils arise, thus under some conditions dissociation of hexamers may be rate limiting or partially rate limiting. The insulin mutants were found to retain substantial nativelike secondary and tertiary structure under all conditions studied. The results suggest that fibril formation of the insulin mutants is controlled by specific molecular interactions that are sensitive to variations in the primary structure. The observed effects of several mutations on the rate of fibril formation are inconsistent with a previously suggested model for fibrillation [Brange, J., Whittingham, J., Edwards, D., Youshang, Z., Wollmer, A., Brandenburg, D., Dodson, G., and Finch, J. (1997) Curr. Sci. 72, 470-476]. Two surfaces on the insulin monomer are identified as potential interacting sites in insulin fibrils, one consisting of the residues B10, B16, and B17 and the other consisting of at least the residues A8 and B25. The marked increase in the lag time for fibril formation with mutations to more polar residues, as well as mutations to charged residues, demonstrates the importance of both hydrophobic and electrostatic interactions in the initial stages of fibrillation. A model for insulin fibril formation is proposed in which the formation of a partially folded intermediate is the precursor for associated species on the pathway to fibril formation.
通过研究20种不同的人胰岛素突变体在低pH值（有利于单体/二聚体的条件）和pH 7.4（有利于四聚体/六聚体的条件）下的原纤维形成的结构特性和动力学，对胰岛素原纤维形成的分子基础进行了研究。小角X射线散射表明，胰岛素在20%乙酸、0.1 M NaCl、pH 2条件下呈单体状态。使用远紫外圆二色性评估突变体的二级结构，使用近紫外圆二色性、丙烯酰胺淬灭内在荧光以及与疏水探针1-苯胺基-8-萘磺酸（ANS）的相互作用来确定三级结构。用荧光染料硫黄素T监测原纤维形成的动力学。结果表明，单体是原纤维产生的状态，因此在某些条件下六聚体的解离可能是限速的或部分限速的。发现在所有研究条件下，胰岛素突变体都保留了大量类似天然的二级和三级结构。结果表明，胰岛素突变体的原纤维形成受特定分子相互作用的控制，这些相互作用对一级结构的变化敏感。观察到的几种突变对原纤维形成速率的影响与先前提出的纤维化模型不一致[Brange, J., Whittingham, J., Edwards, D., Youshang, Z., Wollmer, A., Brandenburg, D., Dodson, G., and Finch, J. (1997) Curr. Sci. 72, 470 - 476]。胰岛素单体上的两个表面被确定为胰岛素原纤维中潜在的相互作用位点，一个由B10、B16和B17残基组成，另一个至少由A8和B25残基组成。随着突变为极性更强的残基以及带电荷的残基，原纤维形成的延迟时间显著增加,这证明了疏水相互作用和静电相互作用在纤维化初始阶段的重要性。提出了一个胰岛素原纤维形成模型，其中部分折叠中间体的形成是原纤维形成途径上相关物种的前体。
