金属硫蛋白及其结构域突变体的紫外和圆二色光谱学研究

采用 p GEX- 4T- 1融合表达载体高效表达所得的金属硫蛋白及其结构域突变体 ,包括 α结构域 ,β结构域 ,α- KKS- α和 β- KKS- β( KKS为金属硫蛋白结构域之间的天然连接区氨基酸 ) ,经纯化和纯度鉴定后 ,利用紫外和圆二色光谱进行结构研究 .在脱金属的上述蛋白中 ,固定 p H为中性 ,改变加入 Cd2 + 的比例 ,或固定 Cd2 + 浓度 ,逐渐调节 p H至中性 ,观察紫外和圆二色光谱中镉硫金属簇吸收峰的形成 .研究结果表明 :镉硫金属簇的形成依赖于加入金属的比例和 p H值 ,所有蛋白均于p H3.1 5以上开始形成明显的吸收峰 .紫外图谱中的吸收峰位于 2 54nm附近 ,但在圆二色图谱中不同蛋白形成的峰的位置不同 ,MT,α结构域和 α- KKS- α在 2 2 5nm和 2 58nm处有吸收 ,β结构域在 2 60 nm处有吸收 ,而 β- KKS- β在 2 4 5nm处有吸收 ;向 α结构域 ,β结构域 ,MT,α- KKS- α和 β-KKS- β中分别加入 4eq,3eq,7eq,8eq和 6eq( eq:equivalent,当量 )的 Cd2 +时 ,吸收峰可达到最大值 .同时发现 α结构域的吸收峰强于 β结构域 ,而且双结构域突变体的镉硫金属簇则明显强于相应的单结构域突变体 ,这表明吸收峰的强弱与金属结合力的大小相关 ,而且结构域之间存在相互作用 ,从而影响与金属的结合 .

Study on the Ultraviolet and Circular Dichroism Spectroscopy of Metallothionein and its Domain Mutants

The metallothionein and its domain mutants including the α domain,β domain,α KKS α,β KKS β(KKS means Lys Lys Ser,which is the linker of α and β domain in native MT),have been expressed with the fusion vector pGEX 4T 1.Having been purified and identified,these protein structures were studied with Ultraviolet spectroscopy and Circular dichroism spectroscopy.The Cd thiolate cluster formation in these proteins have been followed by monitoring UV and CD features as one function of increasing Cd to apoprotein ratio at pH7.5 and the other function of increasing the pH while keeping the Cd 2+ concentration invariable.The spectra showed that the formation of the tetrahedral Cd thiolate cluster depended both on pH and the ratio of Cd to protein.The obvious bands appeared when the pHs of the solution were adjusted over 3.15 for all of the proteins.In the UV spectra,the absorption bands formed near 254nm.While in CD spectra,these proteins showed different ellipticity bands:The ellipticity bands of MT,α domain and α KKS α were characterized at 225 nm and 258 nm;The band of β domain was near 260 nm;The band of β KKS β was near 245 nm.The peaks increased to the maximum when 4eq,3eq,7eq,8eq,and 6eq of Cd 2+ were added to α domain,β domain,MT,α KKS α,and β KKS β respectively.It was found that the absorption band of α domain was stronger than that of β domain;The bands of domain dimers were much more obvious and sharper than that of mono domains.These indicate that the α domain contributed mostly to the spectra of MT,and what's the most important,there was interaction between the two domains which made effect on the metal binding ability,as was also indicated in the UV and CD spectra.