An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins.
An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins.
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DOI：
10.1007/s10858-015-0009-6
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发表时间：
2016-01
影响因子：
2.7
通讯作者：
Subedi GP
中科院分区：
文献类型：
--
作者：
Barb AW;Subedi GP
金属离子在结构生物学应用中发挥着重要作用，从磁共振实验中的远程扰动到x射线晶体学数据中的电子密度特征；然而，金属离子必须被固定在分子框架中才能获得最大的效益。基于多肽的镧系结合标签（lbt）是一种可以直接在重组蛋白表达结构中编码的选择。然而，相对于靶分子，lbt通常表现出显著的移动性。在这里，我们报告了插入蛋白环的改进lbt序列的特性。这些lbt被插入连接免疫球蛋白G （IgG）结合Z结构域平台的两个平行α螺旋。变体A和B结合Tb3+具有高亲和力（分别为0.70和0.13µM），并表现出受限的LBT运动。与亲本结构相比，通过磁场诱导的残余偶极偶联测量，金属结合A的标签运动减少了2.5倍，并在与人IgG1片段结晶（IgG1 Fc）糖蛋白的72.2 kDa复合物中进一步研究。IgG1 Fc:（变体A:Tb3+）2配合物中IgG1 Fc共振的伪接触位移（-0.221至0.081 ppm）和残余偶极耦合（-7.6至14.3 Hz）的出现表明LBT相对于Fc的结构限制。这些研究突出了降低迁移率的改进LBT序列在探测大分子体系结构方面的适用性。
Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in x-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding Z domain platform. Variants A and B bound Tb3+ with high affinity (0.70 and 0.13 µM, respectively) and displayed restricted LBT motion. Compared to the parent construct, the metal-bound A experienced a 2.5-fold reduction in tag motion as measured by magnetic field-induced residual dipolar couplings and was further studied in a 72.2 kDa complex with the human IgG1 fragment crystallizable (IgG1 Fc) glycoprotein. The appearance of both pseudo-contact shifts (-0.221 to 0.081 ppm) and residual dipolar couplings (-7.6 to 14.3 Hz) of IgG1 Fc resonances in the IgG1 Fc:(variant A:Tb3+)2 complex indicated structural restriction of the LBT with respect to the Fc. These studies highlight the applicability of improved LBT sequences with reduced mobility to probe the structure of macromolecular systems.