Folding of Proinsulin

描述(申请人提供)：本申请聚焦于一种具有中心医学重要性的球状蛋白--人胰岛素原--并研究其可折叠性的序列决定因素。在脊椎动物中，胰岛素原的正确折叠对胰岛β细胞的功能和代谢平衡的维持至关重要。 对胰岛素原的基线结构知之甚少，原因是长期以来难以获得晶体，以及其核磁共振谱因聚集而难以处理。在目标1和目标2中，我们将通过对工程单体的异核核磁共振研究来克服这些限制。我们试图将胰岛素部分的结构与胰岛素本身的结构进行比较，并评估连接肽的局部有序程度。目的3研究哺乳动物分泌细胞系内质网可折叠的序列决定因素。这些细胞生物学研究将检验这一假设，即特定折叠核中的残基是正确折叠所必需的，而与突变对折叠蛋白质的稳定性或受体结合亲和力的影响无关。我们假设会发现“折叠突变”阻碍了基态的动力学可及性。目的4以这些结果为基础，验证体内可能存在的折叠突变是否真的会损害胰岛素原的体外氧化折叠。由于使用生物合成材料研究非折叠突变体的结构可能是不可行的，我们建议使用全化学合成来制备相应的突变体胰岛素，用于功能、热力学和结构研究。目标5寻求“可展开”变体的结构。 蛋白质中的特定侧链能否在折叠过程中充当“动力学向导”--但一旦折叠，就可以为蛋白质的功能发挥作用？在噬菌体P22的三聚体尾尖蛋白中发现了经典的折叠途径突变。拟议的研究将测试这一范例是否概括为具有中心药学和生理重要性的单体折叠反应。最令人感兴趣的将是“可折叠”蛋白质的晶体结构。我们的结果有望对胰岛素样序列的进化和人胰岛素原在II型糖尿病中可能的错误折叠产生影响。

DESCRIPTION (provided by applicant): This application focuses on a globular protein of central medical importance - human proinsulin - and investigates sequence determinants of its foldability. Proper folding of proinsulin is fundamental to the function of the pancreatic beta cell and maintenance of metabolic homeostasis in vertebrates. Little is known about the baseline structure of proinsulin due to long-standing difficulties in obtaining crystals and the intractability of its NMR spectrum due to aggregation. In Aims 1 and 2 we will overcome these limitations through heteronuclear NMR studies of an engineered monomer. We seek to compare the structure of the insulin moiety to that of insulin itself and to evaluate the extent of local order in the connecting peptide. Aim 3 investigates sequence determinants of foldability in the endoplasmic reticulum of mammalian secretory cell lines. These cell-biological studies will test the hypothesis that residues in a specific folding nucleus are required for proper folding independently of effects of mutations on the stability or receptor-binding affinity of the folded protein. We hypothesize that "folding mutations" will be found that block kinetic accessibility to the ground state. Aim 4 builds on these results to test whether putative in vivo folding mutations indeed impair oxidative folding of proinsulin in vitro. Because structural studies of nonfolding variants might be infeasible using biosynthetic material, we propose to employ total chemical synthesis to prepare the corresponding mutant insulins for functional, thermodynamic, and structural studies. Structures of "unfoldable" variants are sought in Aim 5. Can specific side chains in a protein serve as "kinetic guides" during folding - but be dispensible for the fuction of the protein once folded? Classical folding-pathway mutations have been identified in the trimeric tail-spike protein of phage P22. The proposed studies will test whether this paradigm generalizes to a monomeric folding reaction of central pharmaceutical and physiological importance. Of overarching interest would be the crystal structure of an "unfoldable" protein. Our results promise to have implications for the evolution of insulin-like sequences and the possible misfolding of human proinsulin in type II diabetes mellitus.