【作者】 刘洪林；

【导师】 韦世强；

【作者基本信息】 中国科学技术大学 ， 同步辐射及应用， 2010， 博士

【摘要】 本论文主要利用同步辐射真空紫外圆二色(SRCD)光谱学方法,结合动态光散射(DLS)、X射线晶体学以及原子力显微镜(AFM)等实验方法研究了溶液条件下生物大分子的结构与相变：(1)原发性痛风病人体内磷酸核糖焦磷酸合成酶1(PRS1)及其点突变体的溶液构象；(2)聚阴离子ATP对溶菌酶解折叠过程的影响；(3)丝素蛋白重链N端亲水区(Fib-H N domain)的结构与功能的关系。在二级结构分析的基础上,分别探讨了点突变导致PRS1超活性的机理、ATP促进溶菌酶形成淀粉样纤维的机制以及Fib-H N domain在自然纺丝过程中发挥的作用。1、点突变导致PRS1超活性的机理本工作表达纯化了PRS1及D52H、N114S、L129、D183H、A190V和H193Q六个致病点突变体,酶活性测试显示点突变导致酶活性显著升高。利用SRCD、DLS等方法研究了它们在结合底物、抑制物前后构象与聚集状态的变化。研究发现PRS1蛋白的活性形式并不依赖于其聚集态,但是底物ATP能够引起PRS1聚集成六聚体。抑制剂ADP结合位点位于六聚结合界面上,所以六聚的形成为ADP进行别构抑制调控提供了结构基础。这一复杂的酶活性调控机制使得酶催化活性处于正常水平。当PRS1发生点突变时,其聚集能力被显著弱化,有的点突变甚至导致聚集消失(比如N114S和L129I),这相应的弱化了ADP的别构调控能力。而且SRCD研究发现点突变导致蛋白底物结合构象改变,晶体结构分析揭示N114S底物结合构象的改变可能会破坏PO43-别构位点。所以点突变导致的底物结合构象的改变也许是酶超活性的另一个原因。这些因素一起导致酶出现超活性,最终引发痛风。2、ATP促进溶菌酶形成淀粉样纤维的机制利用AFM、SRCD、DSC等方法研究了聚阴离子ATP对溶菌酶溶液构象、解折叠过程以及淀粉样纤维形成的影响。研究发现ATP可以结合到溶菌酶上,而且ATP磷酸基团的强烈静电效应使得溶菌酶上暴露的色氨酸残基卷入更加疏水的环境,从而改变了蛋白质的二级结构。在升温解折叠过程中,色氨酸残基位置的改变降低了溶菌酶的热稳定性,导致了二级结构的非协同性展开,在50℃左右产生了一个含有相对丰富螺旋和较少β-片结构的部分展开中间体。DLS实验说明部分展开中间体相比于天然结构具有更强的聚集倾向。此外,ATP非特异性结合到伸展的多肽链上,屏蔽了相应的反应基团,抑制了溶菌酶展开过程的可逆性。因此ATP诱导的溶菌酶的不稳定和非协同性展开,是ATP促进溶菌酶形成纤维状结构的内在基础。同时也表明,相比于单体的富β-片中间体,淀粉样纤维片段的变性程度才是纤维形成的关键因素。3、Fib-H N domain (FN)的构象转变利用SRCD方法研究了四个不同长度的不含信号肽(1～21位残基)区域的FN片段,即FN(22-71)、FN(22-104)、FN(22-126)和FN(22-145),在不同溶液条件下发生的Coil-to-β构象转变,探讨了FN在丝纺织过程中的功能。结合AFM等实验手段,发现在能够形成β-片结构的溶液条件下,这些FN片段能够形成直径为50-100 nm的纳米球状或者纳米囊泡状结构,而且这些纳米球状结构可以互相融合形成不规则的网状结构。并且发现FN片段越长越容易发生构象转变和形成纳米球状结构。相比于丝素蛋白重链中大量的疏水重复区段,Fib-H N domain除了增加蛋白溶解度的功能外,可能扮演着一种感受器的角色,能够迅速感知周围环境改变的信号,从而引起整个丝素蛋白重链的构象变化。这些纳米球状结构的自组装可能作为一个种子启动整个丝素蛋白重链形成纳米原纤维,从而表现出家蚕丝纺织过程中重要的形态学特征。更多还原

【Abstract】 This thesis presents the investigation on the structure and conformational transition of biomacromolecules in solution by synchrotron radiation circular dichroism (SRCD) spectroscopy, combined with X-ray crystallography, dynamic light scattering (DLS), atomic force microscopy (AFM), etc. It involves three different aspects:(1) Identifying point-mutations of human phosphoribosyl pyrophosphate synthetase 1 (PRS1) in cells of patients with primary gout; (2) Observing effects of ATP on the conformation and thermal unfolding of the hen egg white lysozyme (HEWL); and (3) Investigating the conformational transitions of beta sheet proteins: N domain of the silk fibroin heavy chain (Fib-H N domain). The secondary structure analysis for biomacromolecules presented in this thesis is helpful for understanding the structure-biofunction relationship, and additionally demonstrates the valuable applications of SRCD spectroscopy in the biological science.1. Mechanism of PRS1 superactivity caused by the point-mutationsSome cases of point mutation of PRS1 have long been thought to cause PRS1 superactivity, which is one reason of primary gout. In this work, the differences in secondary structure and aggregation state between recombinant wild-type PRS1 and six point-mutations are studied by spectroscopy methods combining with the crystal structure analysis of wild-type PRS1. It is found that the activity of PRS1 does not depend on the subunit-aggregation that has been thought to be necessary for activated state; however, the substrate ATP can induce monomeric wild-type PRS1 to form hexamer which is the structural basis for the ADP binding. Under physiological conditions, the production of ADP downstream is a negative feedback inhibitor that controls the catalytic activity of PRS1 at the normal level. When point mutation occurs, the enzymatic activity dramatically increases, and the aggregation ability of PRS1 is greatly weakened or even lost. Furthermore, SRCD reveals that the point-mutations result in the alteration of ATP-binding conformation. Examination of the crystal structure of wild-type PRS1 indicates that the alteration of ATP-binding conformation can lead to the breakage of the PO43- binding site. Therefore, in the cases of point-mutants, these factors result in an increased concentration of the product PRPP in the patient, and may finally cause gout.2. ATP-induced instability, noncooperative melting and aggregation of HEWL To gain insight into the fibril formation mechanism of proteins, the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light scattering, and DSC, is used to investigate the conformational changes and thermal unfolding of the hen egg white lysozyme (HEWL) in the presence of ATP, ADP, AMP, and Mg2+-ATP complex. The results indicate that the ATP can bind to HEWL, and the strong electrostatic effect of ATP phosphate groups changes tryptophan residues into more hydrophobic environments, and alters the secondary structures of HEWL. This effect decreases the thermal stability of HEWL, induces a noncooperative melting of secondary structures in the unfolding process of HEWL, and sequentially produces a partially unfolded intermediate which contains relatively rich helical structures and lessβ-sheet structures. This study suggests that the extent of denaturation of the amyloidogenic fragments, rather than monomericβ-sheet enriched intermediate, is critical for the fibril formation of proteins.3. Conformational transitions of Fib-H N domain (FN)Four FN segments with different length are prepared for exploring the function of FN in silk spinning process. The SRCD spectroscopy and AFM are used to investigate the conformational and morphological changes of FN in different conditions. This study reveals that FN has the similar structure transition phenomena as regenerated silk functions. All four FN can form the nanoglobular structure with an average diameter dimension ranging from 50 to 100 nm providing that the second structure of the proteins is changed toβ-sheet. These nanoglobular can fuse into each other to form an irregular reticulation. Moreover, the longer FN segment is much easier to have conformational transition and to form the nanoglobular structure. All these results indicate that FN can involve in the silk spinning as a folding promoter instructing the fibroin structure transition.更多还原