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

II类MHC蛋白是a B异二聚体（M.W ~ 66，000）。 的 肽在结合位点被拉伸，形成氢网络 与MHC蛋白质结合。 一些肽侧链 结合部位有很深的口袋 强相互作用 具有结合位点的肽可能在生理上 重要的是，它允许复杂的prestist和增加其 免疫原性 为了获得对MHC II结构的新见解： I-Ak-HEL 48-62，我们探测了肽骨架结合的动力学 通过氢交换动力学与LC-MS/MS组合。 用共价连接的HEL 48-62制备I-Ak蛋白的形式。 蛋白水解后，复合物被一个独特的肽占据。 的 MHC被完全氘化，并且H/D反向交换被启动， 将溶液在pH 7.4的H2O中稀释不同的时间。 的 通过将pH降低至2.75来停止交换。 氘肽 通过快速LC-MS/MS分析研究含量和分布。 提出了一种新的数据处理算法和专用计算机程序， 用来将其他同位素中的氘含量 贡献和计算，从随时间变化的产物离子光谱 氘富集肽的交换速率常数， 结合肽的大部分酰胺键。 的量 交换与对原始人的保护程度有关。 在MHC中的nic肽，并与所看到的非常一致 在晶体结构中。

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.