Role of the linker between the N- and C-terminal domains in the stability and folding of rabbit muscle creatine kinase
Role of the linker between the N- and C-terminal domains in the stability and folding of rabbit muscle creatine kinase
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N-和C-末端结构域之间的连接子在兔肌肉肌酸激酶的稳定性和折叠中的作用
DOI：
10.1016/j.biocel.2007.04.028
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发表时间：
2007-01-01
影响因子：
4
通讯作者：
Yan, Yong-Bin
中科院分区：
文献类型：
--
作者：
He, Hua-Wei;Feng, Shan;Yan, Yong-Bin
结构域-结构域相互作用对于许多多结构域蛋白的结构和功能可能是非常重要的。然而，人们对这种连接体在多结构域蛋白的折叠、稳定性和功能中的作用知之甚少。本研究以二聚体双结构域蛋白肌酸激酶（CK）为模型蛋白，通过突变分析探讨其连接体在CK活性、稳定性和折叠中的作用。三个突变中的两个，L115D和L121D，导致CK活性和二级结构逐渐降低，但不影响热或盐酸胍（GdnHCl）诱导的CK失活。在高温和gdnhcl诱导的变性和gdnhcl变性状态下的重折叠过程中，突变还引起了更严重的聚集。更重要的是，三个突变体都不能成功地通过稀释启动重折叠恢复其活性，并且突变使CK重折叠的速率常数逐渐降低。这些结果表明，连接体中疏水残基的突变可能会影响结构域的正确定位，从而破坏两个结构域之间的有效识别和相互作用。本研究结果表明，除了在体内的功能作用外，该连接子在CK的稳定性和折叠中也起着至关重要的作用。(c) 2007 Elsevier Ltd.版权所有。
Domain-domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of the linker in the folding, stability and function of multidomain proteins. In this research, muscle creatine kinase (CK), a dimeric two-domain protein, was used as a model protein to investigate the role of the linker in CK activity, stability and folding by mutational analysis. Two of the three mutations, L115D and L121D, resulted in a gradual decrease in CK activity and secondary structures, but did not affect CK inactivation induced by heat or guanidine hydrochloride (GdnHCl). The mutations also caused much more serious aggregation during heat- and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, none of the three mutants could successfully recover their activities by dilution-initiated refolding, and the rate constant of CK refolding was gradually decreased by the mutations. These results suggested that mutations of the hydrophobic residues in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the two domains. The results herein indicated that in addition to its role in the in vivo functions, the linker also played a crucial role in the stability and folding of CK. (c) 2007 Elsevier Ltd. All rights reserved.