PROBING BINDING OF ANTIGENIC PEPTIDE TO MHC BY H & D EXCHANGE

H 探测抗原肽与 MHC 的结合

• 批准号: 6665895
• 负责人: EMIL Raphael UNANUE
• 金额: $ 15.75万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6665895
• 关键词: animal tissue; biological products; biomedical resource; endocrine gland /system; enzymes; hormones; liver; spectrometry

Class II MHC proteins are a b heterodimers (M.W ~ 66,000). The peptide is streched in the binding site forming a network of hydrogen bonds with the MHC protein. Some of the peptide side chains interact with deep pockets in the binding site. Strong interaction of the peptide with the binding site is likely to be physiologicaly important, it allows the complex to presist and increase its immunogenicity. To get new insight on the structure of the MHC II: I-Ak-HEL 48-62 we probed the dynamics of the peptide backbone binding by hydrogen exchange kinetics in combination with LC-MS/MS. A soluble form of I-Ak protein was made with a covalently attached HEL 48-62. After proteolysis the complex is occupied by a unique peptide. The MHC is completely deutrated and H/D back exchange is initiated by diluting the solution in H2O at pH 7.4 for varying times. The exchange is stopped by lowering the pH to 2.75. The peptide deuterium content and distribution was investigated by rapid LC-MS/MS analysis. A new data processing algorithm and a dedicated computer program were developed to deconvulute the deuterium content from other isotopic contributions and calculate, from time-dependent product-ion spectra of the deuterium-enriched peptide, the rate constants for exchange at most of the amide linkages of the bound peptide. The amount of exchange is related to the extent of protection afforded the antige nic peptide in the MHC and is remarkably consistent with what is seen in the crystal structure.

II类MHC蛋白是A B杂二聚体（M.W〜66,000）。 这 将肽置于形成氢网络的结合位点 与MHC蛋白结合。 一些肽侧链相互作用 结合位点有深层口袋。 强烈的相互作用 具有结合位点的肽可能是生理的 重要的是，它使综合体能够为 免疫原性。 要获得有关MHC II结构的新见解： i-ak-hel 48-62我们探测了肽主链结合的动力学 通过氢交换动力学与LC-MS/MS结合使用。 可溶性 I-AK蛋白的形式是用共同附着的HEL 48-62制成的。 蛋白水解后，复合物被独特的肽占据。 这 MHC已完全剥离，H/D背面交换由 在不同的时间以7.4的pH值稀释溶液。 这 通过将pH降低到2.75，可以停止交换。 肽氘 通过快速LC-MS/MS分析研究了内容和分布。 一种新的数据处理算法和专门的计算机程序是 开发用于对其他同位素的氘含量去侵入 贡献和计算，从时间依赖性产品离子光谱 依赖氘的肽，即交换的速率常数 结合肽的大多数酰胺连接。 数量 交换与保护范围有关 MHC中的NIC肽，与所看到的非常一致 在晶体结构中。