Kotz Joanne D, Bond Christopher J, Cochran Andrea G
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA.
Eur J Biochem. 2004 May;271(9):1623-9. doi: 10.1111/j.1432-1033.2004.04076.x.
To address questions of protein stability, researchers have increasingly turned to combinatorial approaches that permit the rapid analysis of libraries of protein variants. Phage-display has proved to be a powerful tool for analyzing protein stability due to the large library size and the robustness of the phage particle to a variety of denaturing conditions. With the B1 domain of protein G (GB1) and a camelid heavy chain antibody as model systems, we are using phage-display libraries to experimentally address questions that have generally been addressed in silico, either through computational studies or statistical analysis of known protein structures. One effort has focused on identifying novel solutions to repacking the hydrophobic core of GB1, while maintaining stability comparable to the wild type protein. In a second study, a small set of substitutions in complimentarity-determining region 3 was found to stabilize the framework of the camelid antibody. Another major focus has been to obtain quantitative data on beta-sheet stability determinants. We have successfully adapted a phage-display method for quantitating affinities of protein variants (shotgun alanine scanning) to analysis of GB1 stability. Using this method, we have analyzed the energetic contributions of cross-strand side chain-side chain interactions. Finally, we discuss parameters to consider in using phage-display to discriminate subtle stability differences among fully folded variants. Overall, this method provides a fast approach for quantitatively addressing biophysical questions.
为了解决蛋白质稳定性问题，研究人员越来越多地转向组合方法，这种方法能够快速分析蛋白质变体文库。由于文库规模大以及噬菌体颗粒对各种变性条件具有耐受性，噬菌体展示已被证明是分析蛋白质稳定性的强大工具。以蛋白G的B1结构域（GB1）和骆驼科动物重链抗体作为模型系统，我们正在利用噬菌体展示文库通过实验来解决通常在计算机上通过计算研究或已知蛋白质结构的统计分析来解决的问题。其中一项工作重点是在保持与野生型蛋白质相当的稳定性的同时，找到重新填充GB1疏水核心的新方法。在第二项研究中，发现互补决定区3中的一小部分替换可稳定骆驼科动物抗体的框架。另一个主要重点是获得有关β折叠稳定性决定因素的定量数据。我们已成功将一种用于定量蛋白质变体亲和力的噬菌体展示方法（鸟枪丙氨酸扫描）应用于GB1稳定性分析。使用这种方法，我们分析了跨链侧链 - 侧链相互作用的能量贡献。最后，我们讨论了在使用噬菌体展示来区分完全折叠变体之间细微稳定性差异时需要考虑的参数。总体而言，该方法为定量解决生物物理问题提供了一种快速途径。
