Christian Doppler Laboratory for Antibody Engineering, Vienna Institute of BioTechnology, BOKU University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
J Mol Biol. 2012 Oct 26;423(3):397-412. doi: 10.1016/j.jmb.2012.07.017. Epub 2012 Jul 27.
One of the most important but still poorly understood issues in protein chemistry is the relationship between sequence and stability of proteins. Here, we present a method for analyzing the influence of each individual residue on the foldability and stability of an entire protein. A randomly mutated library of the crystallizable fragment of human immunoglobulin G class 1 (IgG1-Fc) was expressed on the surface of yeast, followed by heat incubation at 79°C and selection of stable variants that still bound to structurally specific ligands. High throughput sequencing allowed comparison of the mutation rate between the starting and selected library pools, enabling the generation of a stability landscape for the entire CH3 domain of human IgG1 at single residue resolution. Its quality was analyzed with respect to (i) the structure of IgG1-Fc, (ii) evolutionarily conserved positions and (iii) in silico calculations of the energy of unfolding of all variants in comparison with the wild-type protein. In addition, this new experimental approach allowed the assignment of functional epitopes of structurally specific ligands used for selection [Fc γ-receptor I (CD64) and anti-human CH2 domain antibody] to distinct binding regions in the CH2 domain.
蛋白质化学中最重要但仍未被充分理解的问题之一是序列与蛋白质稳定性之间的关系。在这里，我们提出了一种分析每个残基对整个蛋白质折叠和稳定性影响的方法。在酵母表面表达可结晶片段的人免疫球蛋白 G 类 1（IgG1-Fc）的随机突变文库，随后在 79°C 下进行热孵育，并选择仍然与结构特异性配体结合的稳定变体。高通量测序允许比较起始和选择文库池之间的突变率，从而在单个残基分辨率下生成人 IgG1 的整个 CH3 结构域的稳定性图谱。使用 IgG1-Fc 的结构、进化保守位置以及所有变体与野生型蛋白相比的展开能的计算进行分析。此外，这种新的实验方法允许将用于选择的结构特异性配体（Fc γ-受体 I（CD64）和抗人 CH2 结构域抗体）的功能表位分配到 CH2 结构域的不同结合区域。
