【作者】 朱振泰；

【导师】 李艳梅；

【作者基本信息】 清华大学 ， 化学， 2005， 博士

【摘要】 脯氨酸在多肽和蛋白的结构和功能中扮演着非常特殊而关键的角色,脯氨酸与其之前的氨基酸残基所形成的肽脯酰胺键在调控生物体内各种生命现象的过程中发挥着类似分子开关的重要作用。影响肽脯酰胺键顺反异构的因素较多,脯氨酸前氨基酸残基的侧链磷酸化是非常重要的一个。研究作为一种重要的翻译后修饰形式的可逆磷酸化对肽脯酰胺键顺反异构的影响,可以为探讨磷酸化对蛋白质结构与功能的影响打下理论基础。本文设计了两类四肽化合物模型,作为研究磷酸化影响肽脯酰胺键顺反异构的框架。分别利用整体法和逐步法,合成了以Ser/Thr/Tyr-Pro片段为研究核心的三个系列的磷肽和非磷肽共计18个。在对合成的模型化合物进行HPLC分离纯化的过程中,通过优化分离条件,初步发现了磷酸化影响肽脯酰胺键顺反异构含量的证据,为建立一种新的顺式构象含量的定量表征方法提供了可能。在对模型化合物的质谱研究中,发现保护氨基酸单体的一个新的经由五员环中间体的ESI-MS碎片重排反应。同时比较了模型化合物的ESI质谱和MALDI-TOF质谱的裂解规律。通过一维核磁手段对模型化合物进行了肽脯酰胺键顺式含量的研究,并以此为基点分别从侧链效应、磷酸化过程本身以及离子效应等方面讨论了磷酸化对肽脯酰胺键顺反异构的影响。通过二维核磁和CD等手段,从构象、空间结构、分子间作用力等方面,探讨了磷酸化对肽脯酰胺键顺反异构产生影响的可能原因。从理论化学的角度,利用分子动力学模拟手段,对磷酸化效应进行了模拟计算,较好地辅证了实验结果。综合所有的研究结果,发现磷酸化对肽脯酰胺键的顺反异构具有影响,磷酸化本身协同电荷效应以及侧链效应共同对肽脯酰胺键的顺式构象含量产生影响,发挥类似分子开关的重要功能。更多还原

【Abstract】 Proline plays an unique and key role in peptide and protein structure. Thecis/trans isomerization of peptidyl-prolyl imide bond formed by proline and theamino acids preceding proline acts as a molecular switch in controlling many lifeprocess in vivo. Among all the factors influencing the cis/trans isomerization, theside-chain O-phosphorylation on the amino acids preceding proline, a well-knownreversible posttranslational modification itself, is important as it might exert directeffects as well as indirect effects on this kind of isomerization. Studies in this fieldmight further illuminate how the phosphorylation regulates the protein function.Two sorts of tetrapeptide models were designed to explore the effects ofphosphorylation on peptidyl-prolyl imide bond cis/trans isomeriztion in this thesis.Phosphopeptides and unphosphopeptides with Ser/Thr/Tyr -Pro motif weresynthesized by global and step-wise solid phase synthesis method based on Fmocstrategy. A new quantitative analysis protocol for the cis content of those peptideswas revealed during the HPLC separation. The MS data for protected amino acidsindicated a new rearrangement reaction via five-membered ring intermediate in ESIfragments. And the fragmentation differences between the ESI-MS andMALDI-TOF-MS were also discussed.Studies on the cis content of all these model compounds were carried out by1H-NMR method. Based on these data, the effects of side-chain, phophorylationprocess and charges were investigated on the cis/trans isomerization of thephosphorylated peptidyl-prolyl amide bond. The possible mechanism were proposed,in the aspects of the conformation, structure and molecular interaction, about theinfluence of phsophorylation on peptidyl-prolyl imide bond cis/transisomerization through 2D NMR and CD studies. Thereafter, the molecularmodeling studies were performed on these model peptides in an attempt to furtherestablish the effect of phsophorylation on the peptidyl-prolyl imide bondcis/trans isomerization and the results were basically consistent with theexperimental data.According to the research, it was concluded that the phosphorylation itself thatcooperated with the side-chain and charge effects influcened the peptidyl-prolylimide bond cis/trans isomerization and subsequently acted as a molecular switch tocontrol the physiological process in life.更多还原