Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
Methods Enzymol. 2021;647:1-22. doi: 10.1016/bs.mie.2020.12.001. Epub 2020 Dec 26.
Peptide linkers consisting of repeats of glycine and serine residues are commonly chosen by protein engineers to introduce flexible and hydrophilic spacers between protein domains. Given the popularity of these linkers, gaining a quantitative insight in their conformational behavior is important to understand the effect on functional properties of fusion proteins, including energy transfer efficiency in luminescent sensor proteins, intramolecular domain interactions and (multivalent) binding. In this chapter, we discuss how the conformational behavior of Ser/Gly linkers can be described using random coil models, and how measuring FRET as a function of linker length can be used to obtain empirical values for the stiffness of linkers containing different Ser-to-Gly ratios. Subsequently, we show how these models and the experimentally determined linker stiffness can be used to explain and predict the functional properties of multidomain proteins, providing useful rules-of-thumb and design tools for optimal linker engineering.
由甘氨酸和丝氨酸残基重复组成的肽接头通常被蛋白质工程师选择，以在蛋白质结构域之间引入柔性和亲水间隔物。鉴于这些接头的普及，定量了解它们的构象行为对于理解融合蛋白的功能特性很重要，包括发光传感器蛋白中的能量转移效率、分子内结构域相互作用和（多价）结合。在本章中，我们讨论了如何使用无规卷曲模型来描述 Ser/Gly 接头的构象行为，以及如何测量 FRET 作为接头长度的函数，以获得具有不同 Ser-to-Gly 比的接头的刚性的实验值。随后，我们展示了如何使用这些模型和实验确定的接头刚度来解释和预测多结构域蛋白的功能特性，为最佳接头工程提供有用的经验法则和设计工具。
