Jacoby E, Hua Q X, Stern A S, Frank B H, Weiss M A
Center on Biomolecular Structure and Function, University of Chicago, IL 60637, USA.
J Mol Biol. 1996 Apr 26;258(1):136-57. doi: 10.1006/jmbi.1996.0239.
The structure and dynamics of the R6 human insulin hexamer are investigated by two- and three-dimensional homonuclear 1H-NMR spectroscopy. The R6 hexamer, stabilized by Zn2+ and phenol, provides a model of an allosteric protein assembly and is proposed to mimic aspects of receptor recognition. Despite the large size of the assembly (36 kDa), its extreme thermal stability permits high-resolution spectra to be observed at 55 degrees C. Each spin system is represented uniquely, implying either 6-fold symmetry or fast exchange among allowed protomeric conformations. Dramatic changes in chemical shifts and long-range nuclear Overhauser enhancements (NOEs) are observed relative to the spectra of insulin monomers. Complete sequential assignment is obtained and demonstrates native secondary structure with distinctive R-state N-terminal extension of the B-chain alpha-helix (residues B1 to B19). The distance-geometry structure of an R-state promoter is similar to those of R6 crystal structures. Specific long-range intra- and intersubunit NOEs, assigned by stepwise analysis of engineered insulin monomer and dimers, demonstrate that tertiary and quaternary contacts are also similar. Although the hexamer is well-ordered in solution, binding of phenol to an internal cavity occurs within milliseconds, implying the existence of "gatekeeper" residues whose flexibility provides a portal of entry and release. Changes in 1H-NMR chemical shifts on hexamer assembly are readily rationalized by analysis of aromatic ring-currents and provide sensitive probes for sites of protein-protein interaction and phenol binding. Our results provide a foundation for the interaction and phenol binding. Our results provide a foundation for the studies of insulin analogues (such as "designed" insulins of therapeutic interest) under conditions of clinical formulation and for the investigation of the effects of protein assembly on the dynamics of individual protomers.
通过二维和三维同核1H-NMR光谱研究了R6人胰岛素六聚体的结构和动力学。由Zn2+和苯酚稳定的R6六聚体提供了一个变构蛋白组装模型，并被认为模拟了受体识别的某些方面。尽管该组装体尺寸较大（36 kDa），但其极高的热稳定性使得在55℃时仍能观察到高分辨率光谱。每个自旋系统都有独特的表示，这意味着要么具有六重对称性，要么在允许的原体构象之间快速交换。相对于胰岛素单体的光谱，观察到化学位移和远程核Overhauser增强（NOE）有显著变化。获得了完整的序列归属，并证明了具有独特的B链α-螺旋R态N端延伸（残基B1至B19）的天然二级结构。R态启动子的距离几何结构与R6晶体结构相似。通过对工程化胰岛素单体和二聚体的逐步分析确定的特定远程亚基内和亚基间NOE表明，三级和四级接触也相似。尽管六聚体在溶液中排列有序，但苯酚在几毫秒内就会与内部腔室结合，这意味着存在“守门人”残基，其灵活性提供了进入和释放的通道。通过对芳香环电流的分析，很容易解释六聚体组装时1H-NMR化学位移的变化，并为蛋白质-蛋白质相互作用和苯酚结合位点提供了灵敏的探针。我们的结果为相互作用和苯酚结合提供了基础。我们的结果为在临床制剂条件下研究胰岛素类似物（如具有治疗意义的“设计”胰岛素）以及研究蛋白质组装对单个原体动力学的影响奠定了基础。
