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

第二类MHC蛋白为a、b异源二聚体，分子量在66,000之间。这个 多肽被张开在结合部位，形成一个氢网络 与MHC蛋白结合。一些多肽侧链相互作用 在结合部位有很深的口袋。的强交互作用 多肽与结合部位很可能是生理学上的 重要的是，它允许建筑群防止和增加其 免疫原性。为了对MHC II的结构有新的了解： I-AK-HEL 48-62探讨了多肽骨架结合的动力学 氢交换动力学与LC-MS/MS联用 用共价连接的HEL 48-62制备I-AK蛋白。 蛋白质分解后，该复合体被一种独特的多肽占据。这个 MHC完全去中性，H/D反向交换由 将溶液在pH为7.4的水中稀释不同时间。这个 通过将pH降至2.75来停止交换。重离子多肽氘 采用LC-MS/MS快速分析方法对其含量和分布进行研究。 提出了一种新的数据处理算法和专用计算机程序 开发用来从其他同位素中分离出氢的含量 贡献，并根据随时间变化的产物离子光谱进行计算 在富氚多肽中，交换速率常数 结合多肽的大部分酰胺键。金额的多少 交换与提供的保护范围有关。 MHC中的NIC多肽，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.