Thermodynamic model for the stabilization of trigonal thiolato mercury(II) in designed three-stranded coiled coils
Thermodynamic model for the stabilization of trigonal thiolato mercury(II) in designed three-stranded coiled coils
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DOI：
10.1021/bi015649a
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发表时间：
2001-12-04
期刊：
影响因子：
2.9
通讯作者：
Pecoraro, VL
中科院分区：
文献类型：
--
作者：
Farrer, BT;Harris, NP;Pecoraro, VL
本文建立了一个热力学模型，描述了Hg（II）与Tri家族衍生的新设计肽Tri L9C和Baby L9C的结合。三肽基于亲本序列Ac-NH-G(LKALEEK)(x)G-CONH2，已知在低pH （pH 7）下形成双链卷曲线圈。Tri L9C （x = 4）包含4个七核苷酸重复序列，其中半胱氨酸位于9位，亮氨酸位于其他a和d位；婴儿L9C (x = 3)，在位置9也有半胱氨酸，但比Tri L9C短一个七格，被设计成在溶液中形成不太稳定的螺旋线圈。Tri, Tri L9C，形成线圈的自由能。用胍变性滴定法测定pH为2.5和8.5时的Baby Tri和Baby L9C；在pH为8.5时，在不添加变性剂的情况下，Tri L9C呈高螺旋状，而Baby L9C在pH为8.5时螺旋状含量< 20%，表明其呈弱相关或不相关的螺旋状。粒径排除色谱法（SEC）证实，在pH为8.5时，Baby L9C为单体。添加HgCl2诱导了Baby L9C的螺旋度。在248nm处，HgS3紫外波段的存在证实了在三股线圈内部形成的三角硫原子汞（II）。将Tri L9C和Baby L9C滴定到pH值在7到9之间的HgCl2溶液中，以提取结合常数。对数据的整体拟合采用了一种机制，该机制涉及汞与肽的初始结合，形成与线性硫代汞（II）以[peptide]/[Hg] 7.5 （Hg（II）存在时半胱氨酸硫醇的pK(a)）的双链盘绕线圈。在ph7.0的Tri L9C溶液中，用SEC对三股线圈的观察证实了这一机理。
A thermodynamic model is presented that describes the binding of Hg(II) to de novo designed peptides, Tri L9C and Baby L9C, which were derived from the Tri family. The Tri peptides are based on the parent sequence Ac-NH-G(LKALEEK)(x)G-CONH2 and are known to form two-stranded coiled coils at low pH (pH 7). Tri L9C (x = 4) contains a four heptad repeat sequence with cysteine in position 9 and leucines in the other a and d positions; Baby L9C (x = 3), which also has a cysteine in position 9 but is one heptad shorter than Tri L9C, was designed to form less stable helical coiled coils in solution. The free energies of coiled coil formation for Tri, Tri L9C. Baby Tri, and Baby L9C at pH 2.5 and 8.5 were determined by guanidinium denaturation titrations; Tri L9C was observed to be highly helical in the absence of denaturant at pH 8.5 while Baby L9C contained < 20% helical content at pH 8.5, indicating a weakly associated or unassociated coiled coil. Size-exclusion chromatography (SEC) verified that Baby L9C was a monomer at pH 8.5. The helicity of Baby L9C was induced by addition of HgCl2. The subsequent formation of a trigonal thiolato Hg(II) in the interior of a three-stranded coiled coil was verified by the presence of a characteristic HgS3 UV band at 248 nm. Titrations of Tri L9C and Baby L9C into solutions of HgCl2 at pH values between 7 and 9 were performed to extract binding constants. Global fits to the data employed a mechanism that involved initial binding of mercury to the peptides forming a two-stranded coiled coil with linear thiolato Hg(II) at [peptide]/[Hg] 7.5 [the pK(a) of the cysteine thiol in the presence of Hg(II)]. Support for this mechanism was given by the observation of a three-stranded coiled coil by SEC in a solution of Tri L9C at pH 7.0.