【作者】 刘主；

【导师】 唐淳；

【作者基本信息】 中国科学院研究生院（武汉物理与数学研究所） ， 分析化学， 2014， 博士

【摘要】 泛素(Ub:ubiquitin)是真核细胞中非常重要的一类调控蛋白,它几乎参与了.所有的生理过程。Ub通过保守的作用界面、以不同的构象结合不同的靶蛋白,调控细胞内一系列的下游信号传递过程。那么泛素是如何通过这样一个保守的作用界面实现与诸多不同靶蛋白的相互作用,从而行使它复杂而又多样的生理功能的呢?目前为止,答案还不确定。此外,Ub在参与多样的生理过程中,其重点和复杂性还在于它可以形成多聚体。Ub的碳端(distal亚基)可以共价的链接到另外一个Ub的氮端或者赖氨酸残基(Lys)的侧链氨基(proximal亚基)上面,形成以异肽键链接的二聚体。其中K63位链接的Ub二聚体K63-diUb被认为是最“经典”的多聚形式,它与DNA的损伤修复以及先天性细胞免疫相关。目前为止,已被报道的K63-diUb的四级结构都是打开的构象,其闭合态的结构还未得到解析。K63-diUb是否存同时存在打开态与闭合态,两者之间是否能够动态的相互转换,它与靶蛋白的相互作用机制如何?这些问题都还有待进一步的研究。顺磁弛豫增强核磁共振技术(PRE:paramagnetic relaxation enhancement)可以提供长程距离信息用于蛋白质结构的研究。由于PRE对距离的变化非常敏感,它可以独特的用来研究和表征蛋白质低分布的瞬态结构以及弱的相互作用,揭示蛋白质不同构象之间的相互转化过程。因此,PRE技术被越来越广泛的应用于蛋白质动态结构和功能的研究。因此,本论文应用基于PRE的研究方法对Ub,以及Ub二聚体K63-diUb四级结构的动态变化和功能进行了系统的研究；为了描述Ub更精细的动态结构和功能,我们发展了一种刚性标记的PRE探针；此外,我们运用PRE的研究手段进一步的对其它重要蛋白质的动态结构和功能进行了初步研究。我们的研究首次发现,Ub存在低分布的非共价二聚体,KD～5mM,其作用界面分布在Ub的β-折叠区域。该界面和Ub与诸多靶蛋白相互作用的保守界面重叠,因此我们推测,Ub可以通过自身单体-二聚体的平衡来调控它与靶蛋白的相互作用。依据PRE的距离约束,我们解析了Ub非共价二聚体系综结构,这些分布广泛的空间构象涵盖了多种已经解析的Ub共价二聚体的结构。我们的研究表明,Ub非共价的二聚相互作用广泛的存在于Ub共价二聚体中,后者亚基间的相互作用调控Ub共价二聚体的四级结构,使得Ub共价二聚体呈现丰富的空间构象。基于Ub单体动态结构的研究基础,我们进一步的研究了K63-diUb四级结构的动态变化,及其与靶蛋白相互作用的分子机制。采用PRE的研究手段,我们表征了K63-diUb分子内亚基间的相互作用,说明它同时存在闭合态与打开态,其比例分别是80%和20%；首次报道了K63-diUb闭合态的系综结构,结合PCA的分析我们发现闭合态有两种：C1和C2,其比例分别为60%和20%；K63-diUb的三个态之间能够相互转换,它与靶蛋白的结合强度与相应态的比例正相关。因此,K63-diUb可以通过这种态之间的动态转换,以构象选择的结合机制识别不同的靶蛋白,从而调控不同的细胞泛素信号通路,参与不同的生理过程。本论文发展了一种刚性标记的PRE探针diHis-Cu2+-cap用于Ub和其它蛋白质更精细的动态结构的研究。我们发现,Ub残基52-53和55-56所在loop存在低分布的瞬态结构,该loop以残基54为中心前后扭动,其扭动幅度很小,大约2A。此外,采用这种刚性探针标记的PRE,我们发现,holo态的谷氨酰胺结合蛋白(holo QBP) F13残基所在loop也存在细微运动,平均运动幅度只有0.6A,但是它的运动参与配体谷氨酰胺的解离,该loop背离谷氨酰胺的细微运动使得后者的解离成为可能。最后,我们进一步的运用PRE的研究手段对另一类重要蛋白质：谷氨酸受体(GluA2)的脱敏机制进行了初步的研究。初步结果表明,GluA2的脱敏速率与其配体结合结构域(LBD)的二聚相互作用强度相关：二聚相互作用越强,脱敏越慢；二聚相互作用越弱,则脱敏越快。综上所述,本论文通过对Ub,以及Ub二聚体K63-diUb动态结构和功能的系统性研究,从分子层次的水平上阐述了它们四级结构之间的相互转换过程,揭示了它们参与细胞泛素信号传递的分子机制,为实现细胞泛素信号通路的控制提供了结构基础和数据参考。就PRE的方法学而言,我们发展的刚性顺磁探针拓宽了PRE的研究内容,在后续的研究过程中,PRE将解答更多的生物学问题。更多还原

【Abstract】 Ubiquitin (Ub) is a significant regulatory protein in eukaryote cells, and is involved in almost every physiological process. Ub interacts with myriad partner proteins to fulfill diversely functions; however, the interactions invariably involve a hydrophobic patch surrounding residue144. So how does Ub recognize a specific partner protein through such a conserved binding interface to regulate different cell signaling pathway?Furthermore, the complexity of Ub involved in physiological process is that Ub functions mostly as poly-ubiquitin chains. Two or more Ub molecules can be covalently linked via an isopeptide bond between the C-terminal carboxyl group of one Ub (dsital unit) and a primary amine of a lysine residue or N-teminal methionine in another one(distal unit). Lys63-lingked dimmer Ub (K63-diUb) is considered as a "typical" dimmer that is involved in cellular events like DNA damage response and innate cell immunity upon binding to a specific partner protein. Open conformation of ligand-free K63-diUb(apo K63-diUb) was characterized by many biophysical techniques; however, no closed conformation has been captured yet. Does apo K63-diUb exist in both open and closed states and whether there is a dynamic equilibrium between different states? Do these different conformational states could be interconverted? How does K63-diUb recognize and bind to a specific partner protein? Much more detailed descriptions of K63-diUb dynamics and conformational apace are highly deserved, and the connection between conformational dynamics and targets biding events should be addressed.Paramagnetic relaxation enhancement (PRE) affording long-range distance restraints favor protein structure determination. PRE is very sensitively to a distance change because of distance-dependence between paramagnetic center and protein nuclei, on a<r-6> magnitude. As a result, PRE could be used especially to characterize lowly-populated transient protein structures and ultra-weak protein-protein interactions.Here, we integrate PRE and other biophysical techniques and analysis methods to characterize the quaternary dynamics of Ub and K63-diUb. A rigid paramagnetic probe for PRE measurement is engineered to visualize subtle dynamics of Ub or other protein system. Preliminary dynamics and functional investigation of another important protein is also studied. In this study, we show that Ub dimerizes noncovalently through an interface around the β-sheet region with a KD of about5mM. The noncovalent dimmer interface overlaps with the conserved binding interface of Ub to target protein. Thus, the binding events between Ub and target protein probably could be regulated through the Ub monomer-dimmer dynamic equilibrium. The ensemble structures of Ub noncovalent dimmer adopt a wide range of relative subunit orientations, and some conformers are compatible with certain covalent linkage dimmer. As such, the covalent linkage in an Ub dimier may further promote noncovalent Ub-Ub interactions and favor a subset of the ensemble conformation, which would then tailor the interactions with a specific target protein.Based on the dynamics information of mono Ub, we resort to conjoined use of PRE and principal component analysis (PCA) to visualize conformational space of K63-diUb involved in target recognition. We show that K63-diUb adopts three conformational states, comprising one open state and two closed states, namely Cl and C2, with characteristic Boltzmann distribution—20%in open state,60%in Cl, and20%in C2. The three conformational states exist in dynamic equilibrium and can be readily interconverted. Importantly, each conformational state encompasses fhe known structures of K63-diUb with a particular ligand bound, and the respective binding affinity is weighted by the population of the preexisting conformational state. Thus, K63-diUb could recognize specific target protein through conformational selection mechanism based on the dynamics equilibrium and population shift between mutiple conformational states to regulate cell signaling and involved in different physiological process.In order to reveal subtler dynamics of Ub or other protein system, a rigid paramagnetic probe—diHis-Cu2+-cap is engineered. Attached with this rigid paramagnetic probe, PRE reveals that a flexible loop of Ub undergoes a collective pincer-like movement about2A on average, comprising residues52-53and55-56wobbled around residue54. In another protein system, holo glutamine binding protein (holo QBP), subtle dynamics (about0.6A on average) is also revealed with the rigid paramagnetic probe. This loop, comprising residue F13, packs against the glutamine ligand in the holo QBP. As such, the loop movement away from the ligand may facilitate the ligand dissociation.Last, PRE is also applied to reveal the desensitization mechanism of glutamate acceptor A2(GluA2). Preliminary results show that the desensitization rate is coupled with the dimeric strength of the ligand binding domain (LBD). Dimeric interactions stronger, GluA2desensitize slower; dimeric interactions weaker, then faster desensitization.In summary, this thesis work mainly characterize the dynamics of Ub and K63-diUb at quaternary level, and offer more understanding of protein function by detailed description of protein conformational space and protein dynamic equilibrium. Rigid paramagnetic labeling strategy and probe broaden the research content of PRE, and PRE will offer much more information in biological issues.更多还原