【作者】 刘东升； 谢韬； 冯银刚； 单璐； 王金凤；

【Author】 Dongsheng Liu Tao Xie Yingang Feng Lu Shan Jinfeng Wang (National Laboratory of Biomacromolecules,Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China)

【机构】 中国科学院生物物理研究所生物大分子国家重点实验室；

【摘要】 金黄色葡萄球菌核酸酶 N 末端110片段(SNase110),包含残基1-110,处于去折叠态和折叠态之间的动态平衡中,且主要以去折叠态存在。加入氧化三甲基胺(TMAO,Trimethylamine N-oxide),可以使 SNase110片段的折叠形成协同稳定的β桶结构。G88W110片段包含核酸酶1-110序列,其88位的甘氨酸被色氨酸替代,V66W110片段包含核酸酶1-110序列,其66位的缬氨酸被色氨酸替代。由于色氨酸的稳定作用, 它在生理条件下折叠成稳定协同的结构,其结构与核酸酶天然态结构中相应部分十分相似。G88W 和 V66W 突变主要通过增加β桶的折叠稳定性而 TMAO 主要通过增强蛋白主链与溶液的增溶作用来使蛋白质趋于折叠。运用远紫外及近紫外圆二色谱、内源荧光光谱和核磁共振光谱等方法确定了 GB8W110,V66W110和 SNase110片段在2．0 M TMAO 浓度下的去折叠自由能、折叠构象和内运动。用异核多维核磁共振方法,计算了三个片段的溶液三维结构,它们都形成了稳定的β桶结构,α螺旋也初步形成,但都存在一段无序的结构。G88W110的结构最紧凑,而 V66W110结构比较松散。G88W110,V66W110和在2．0 M TMAO 中的 SNase110显示出不同的主链弛豫性质,突变和渗透质对蛋白结构和运动性的影响不同,不同的折叠程度反映出不同的弛豫性质,而 ms-μs 时间的运动可能与折叠的起始位点有关。主链动态特性和脲变性曲线都表明三个片段处于不同的折叠状态．片段的结构和稳定性与β桶结构中β链的堆积程度紧密相关,β桶和蛋白整体结构的形成是偶连的,三个片段的折叠过程是高度协同的。更多还原

【Abstract】 This study investigates the folding stability and cooperativity in the three forms of 1-110 residues fragment of staphylococcal nuclease(SNase110),namely G88W110 and V66W110 in aqueous solution and SNase110 in 2.0 M TMAO.The unfolding free energies,folding conformations,and internal motions of these fragments were determined by far-and near-UV CD,intrinsic tryptophan fluorescence,and NMR spectroscopy.The fragments are found to be in different folding states having different folding stabilities and dynamic properties.Both Gggw and V66W single mutation as well as a small osmolyte(TMAO)can fold the fragment into a native-like conformation.G88W110 adopts a relatively rigid structure representing a most stable native-like β-subdomain conformation of the three fragments.V66W110 and TMAO stabilized SNase110 produce unstable structures having an unstable“β-barrel”structural region in the tertiary conformation.The different folding status accounts for the different backbone dynamic and urea-unfolding transition features displayed by the three fragments.The structure and stability of the fragments are correlated closely to the packing of the β-strands in the“β-barrel”,which plays a key role in the formation of a stable structure of the fragment.The G20I/G29I mutant variants of the three forms 110-residues SNase fragments provide the strong support for it.The formation of“β-barrel”and overall structure is coupled and the folding process is highly cooperative in the three 110-residue SNase fragments.更多还原