Folding of Proinsulin

描述（由申请人提供）：本申请关注具有重要医学意义的球状蛋白-人胰岛素原-并研究其折叠性的序列决定因素。胰岛素原的正确折叠对于胰腺β细胞的功能和维持脊椎动物的代谢稳态是至关重要的。 由于长期难以获得晶体以及由于聚集导致的NMR谱的棘手性，对胰岛素原的基线结构知之甚少。在目标1和2中，我们将通过工程单体的杂环NMR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，并评估连接肽中的局部顺序的程度。目的3研究哺乳动物分泌细胞系内质网折叠性的序列决定因素。这些细胞生物学研究将检验这样的假设，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的稳定性或受体结合亲和力的影响无关。我们假设，“折叠突变”将被发现，块动力学可访问的基态。目的4建立在这些结果的基础上，以测试是否推定的体内折叠突变确实会损害体外胰岛素原的氧化折叠。由于非折叠变体的结构研究可能是不可行的，使用生物合成材料，我们建议采用全化学合成制备相应的突变胰岛素的功能，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在折叠过程中是否可以作为“动力学向导”--但一旦折叠，蛋白质的功能就可以不需要了？在噬菌体P22的三聚体尾刺蛋白中已经鉴定出经典的折叠途径突变。拟议的研究将测试这种模式是否推广到中央药学和生理重要性的单体折叠反应。最令人感兴趣的是“不可折叠”蛋白质的晶体结构。我们的研究结果有望对胰岛素样序列的演变和II型糖尿病中人胰岛素原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.