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肌酸激酶结构域相互作用与活性中心极性微环境的研究
Studies of the Domain-domain Interactions and Polar Microenvironment Around the Active Sites of Creatine Kinase
【作者】 何华伟;
【作者基本信息】 清华大学 , 生物学, 2007, 博士
【摘要】 很多包含有多个结构域的蛋白,其每一个结构域都被认为是结构、折叠、进化和功能的基本单元。多结构域蛋白的折叠一般认为是有序的,每个结构域具有不同的稳定性,它们独立自主地进行折叠。然而,多结构域蛋白要比单结构域蛋白的折叠机制复杂得多,因为它既包含了单个结构域的折叠,同时还有结构域之间的相互作用。当结构域之间存在相互作用时,结构域的折叠和组装对结构域或蛋白总体的稳定性具有重要的意义,甚至对它们在体内的功能产生影响。除此之外,结构域之间的连接序列对蛋白结构的稳定、折叠途径和功能也有重要的影响。然而,人们对此却知之甚少。人们发现肌酸激酶热聚沉发生温度(约47℃)远低于其结构去折叠时的融化温度(约56℃),然而,这种热诱导的蛋白快速失稳和聚沉机制人们并不清楚。本文用定量二阶远红外分析和二维远红外光谱分析方法,结合圆二色和内源荧光光谱研究了肌酸激酶热失活和聚沉过程中的顺序事件,并研究了C末端结构域对肌酸激酶结构稳定性的影响和在肌酸激酶折叠过程中的作用,提出C末端结构域的构象变化启动了蛋白的热聚沉。肌酸激酶结构域之间的连接片段没有形成任何常规的二级结构,其结构具有较大的柔性,而且它主要由带电荷的残基组成,其中仅有四个疏水性的保守残基。本文通过突变和光谱学分析,发现连接片段和C末端结构域之间的疏水相互作用对肌酸激酶活性、稳定性和折叠具有重要意义。连接片段的定位可以帮助肌酸激酶两个结构域正确组装成为具有活性的天然结构。蛋白的极性对其结构、催化和功能都有重要的意义。在本文中,我们选取5,5 ?-dithiobis (2-nitrobenzoic acid)、6,6 ?-dithiodinicotinic acid和2,2 ?-dithiodipyridine研究了肌酸激酶活性中心附近的Cys283周围的极性微观环境。我们研究了三个修饰试剂对肌酸激酶在底物存在下的完整的失活动力学,并获得了游离酶和酶-底物复合物相对应的微观速率常数。结果表明肌酸激酶活性位点附近的Cys283周围主要是一种亲负电性的极性微环境,并且这种微环境很可能就是造成其pKa值较低的原因。
【Abstract】 Many large proteins are composed of domains that can be regarded as structure, function, evolution and folding units. For these multidomain proteins, folding is generally thought to be hierarchical and the individual domains with different stabilities fold autonomously and independently. However, the folding mechanism of multidomain proteins may be much more complicated than single-domain proteins because it involves both the folding of the individual domains and the docking of these domains. Domain folding and assembly play a role in the stability of the domains or the whole protein, even for their functions in vivo, when domain-domain interactions is present. The domains are connected sequentially by the linker in the primary structure. However, little is known about the role of the linker in protein folding, stability and function.The aggregation occurred at a temperature (about 47℃) far below the Tm (about 56℃) of creatine kinase. However, the mechanism of the fast destabilization and aggregation of creatine kinase induced by heat is not clear yet. In this research, the sequential events of creatine kinase thermal inactivation and aggregation were studied by quantitative second derivative infrared analysis and two-dimensional infrared correlation spectroscopy, combined with circular dichroism spectroscopy and tryptophan fluorescense spectroscopy. The results indicated that C-terminal domain played an important role in the structural stability and folding of creatine kinase. We presented a possible mechanism of creatine kinase thermal inactivation and aggregation and suggested that the conformational change in the C-terminal domain was responsible for the initiation of creatine kinase thermal aggregation.The domains are connected sequentially by the linker in the primary structure. The linker does not form any regular secondary structures and seems to be flexible. Moreover, the linker is mainly composed of charged residues and only contains four hydrophobic conserved residues. In this research, the results suggested that the hydrophobic interactions between the linker and the C-terminal domain played a key role on the activity, structural stability and folding of creatine kinase by mutation and spectral analysis. The location of the linker sequence may be helpful to the natvie, active conformation formation assemble by the two domains of creatine kinase correctly.The polarities of protein have an important role in the structures, catalysis and functions. In this research, 5,5 -?dithiobis(2-nitrobenzoic acid), 6,6 -?dithio- dinicotinic acid and 2,2 -?dithiodipyridine were used as specific sulphydryl reagents to explore the polar microenvironment around Cys283, a conserved amino acid nearby the active site of creatine kinase. Complete kinetics of inactivation was recorded, and the relevant microscopic rate constants of the reagents with various enzyme-substrate complexes were also determined. The results indicated that there may exist a polar microenvironment which is predominant by the electropositive residues and this may be the reason for the unusually low pKa value of Cys283.
【Key words】 creatine kinase; domain; linker sequence; protein polarity; active site;