【作者】 刘飞；

【导师】 汪劲；

【作者基本信息】 吉林大学 ， 理论物理学， 2017， 博士

【摘要】 考虑到理论上研究蛋白质生物化学过程中的动力学和热力学对理解其生物功能和本质及其重要,而实验上所能给出的测量通常是宏观的热力学或者动力学量,缺乏在分子层次上理解生命附加活动的过程。本文主要研究兴趣是:以分子动力学模拟为基础手段,建立起一套可以捕捉到某些由于实验精度限制而不能观测到的蛋白质生物学过程的模拟方法,在分子层次上去理解这些生物学过程。从而实现理论和实验桥梁的作用,深化对生物蛋白分子功能的理解。1.钙离子钙调蛋白能(Ca M)与超过300多种的目标蛋白发生结合。探索钙离子钙调蛋白与不同目标蛋白结合的分子机制对理解蛋白质之间的多特异性结合是至关重要的。为了系统的解决这一问题,我们开发分子动力学模拟模型来解决这一问题。我们首先选取了骨骼肌肌球蛋白轻链激酶(sk MLCK)作为钙调蛋白的结合靶标。sk MLCK结合肽有26个残基。我们建立了粗粒化的基于结构的模型。我们发现静电和疏水这两种不同的非天然相互作用在Ca M和其目标的非特异性结合中起着重要的作用。我们也发现了保守性疏水锚在Ca M与其结合目标的过程中的关键性的作用。尤其是结合目标疏水锚和Ca M丰富的MET残基之间的作用在高亲和性到多特异性转化过程中起着决定性的作用。尤其,我们发现了Ca M与其结合目标采用的是非典型的“诱导契合”,非典型的“构象选择”,“同时折叠结合”三者混合的结合机制。在此基础上,我们把讨论扩展到了Ca M与结合目标的多特异性结合上,提出了决定钙调蛋白与其靶标具有多特异性结合特性的几点重要的原因。2.蛋白质和DNA之间的识别作用对细胞生命是至关重要。我们发展了双势阱基于结构的模型来捕获DPO4在与DNA结合过程中自身的构象转变。我们的结果清楚的展现了DPO4和DNA的识别过程包括,三维扩散,在DNA上面的短程滑动和最后的特异性识别这三个过程。同时,DPO4的构象动力学发生在于DNA结合的过程中,且不同的阶段其构象的分布情况有很大的不同。这说明了DPO4的构象被DNA结合所调整。更微观的,我们发现静电作用在三维扩散过程中充当着“驱动力”的作用,相反的,其会通过形成非天然动力学势阱的方式阻碍DPO4在DNA上面的短程滑动。DPO4与DNA结合的效率被这几个结合过程之间交替相互作用所决定。尤其,我们发现T域和F域之间的带负电荷的linker的柔性对DPO4的构象的分布和非特异复合物的稳定性都起着关键的作用。因此,linker对DPO4和DNA的识别也是关键的。总之,我们的模型通过显性的考虑DPO4和DNA之间的非特异性和特异性相互作用,为DPO4与其目标DNA结合的过程提供了一个完整的描述。我们的工作也能为理解伴随着构象转变蛋白质DNA特异性识别提供启示,也丰富了我们对DNA聚合酶催化机制的理解。更多还原

【Abstract】 It is very important to explore the dynamics and thermodynamic theory of biological process.As constrain of resolution of experiment,the exploration of biological process in molecular level is lacking.In this paper,based on the method of molecular dynamic simulation,we develop a systematic theory to explore the interesting and representative biological process which can’t be captured by the experiment.Bridging between theory and experiment,the function of molecule can be uncovered in higher level.1.Calmodulin(CaM)is found to have the capability to bind multiple targets.Investigations on the association mechanism of CaM to its targets are crucial for understanding protein–protein binding and recognition.Here,we developed a structure-based model to explore the binding process between CaM and skMLCK binding peptide.We found the cooperation between nonnative electrostatic interaction and nonnative hydrophobic interaction plays an important role in nonspecific recognition between CaM and its target.We also found that the conserved hydrophobic anchors of skMLCK and binding patches of CaM are crucial for the transition from high affinity to high specificity.Furthermore,this association process involves simultaneously both local conformational change of CaM and global conformational changes of the skMLCK binding peptide.We found a landscape with a mixture of the atypical “induced fit,” the atypical “conformational selection,” and “simultaneously binding–folding,” depending on the synchronization of folding and binding.Finally,we extend our discussions on multispecific binding between CaM and its targets.These association characteristics proposed for CaM and skMLCK can provide insights into multispecific binding of CaM。2.Protein-DNA recognition is a central biological process that governs the life of cells.Here,we performed thermodynamic and kinetic simulations to explore the conformational transitions of DPO4 during binding to the target site in DNA by developing a structure-based model.Our results clearly showed that DPO4-DNA recognition involves 3D diffusion,then a short-range adjustment sliding on DNA and finally specific binding.The conformational changes in DPO4 happen throughout the binding process,with different stages representing different conformational distributions.The conformational dynamics in DPO4 seem to be fine-tuned by DNA.Meanwhile the different conformations of DPO4 have different effects on the DNA binding kinetics.We also found that electrostatic interactions on one hand facilitate the 3D diffusion as “steering forces”,and on the other hand hinder short-range sliding on DNA due to formation of non-native kinetic traps.The efficiency of specific DPO4-DNA recognition is determined by the interplay between multiple binding stages.In particular,we point out that the flexibility of the positively charged linker not only contributes to the conformational distribution in DPO4,but is also responsible for the stabilization of the non-specific complex,and is therefore critical for DPO4-DNA recognition.Our methods,with explicit consideration of the non-specific and specific interactions between DPO4 and DNA,provide a detailed description of the process of DPO4 binding to its target DNA.This work illustrates the process of specific protein-DNA recognition and the accompanying conformational dynamics,and thus enriches our understanding of the catalytic mechanism of DNA polymerization.更多还原