Department of Chemistry , University of Zurich , Zurich 8057 , Switzerland.
Biomolecular Dynamics, Institute of Physics , Albert Ludwigs University , Freiburg 79104 , Germany.
J Phys Chem B. 2018 Nov 8;122(44):10118-10125. doi: 10.1021/acs.jpcb.8b08368. Epub 2018 Oct 30.
The noncanonical amino acid azidohomoalanine (Aha) is known to be an environment-sensitive infrared probe for the site-specific investigation of protein structure and dynamics. Here, the capability of that label is explored to detect protein-ligand interactions by incorporating it in the vicinity of the binding groove of a PDZ2 domain. Circular dichroism and isothermal titration calorimetry measurements reveal that the perturbation of the protein system by mutation is negligible, with minimal influence on protein stability and binding affinity. Two-dimensional infrared spectra exhibit small (1-3 cm) but clearly measurable red shifts of the Aha vibrational frequency upon binding of two different peptide ligands, while accompanying molecular dynamics simulations suggest that these red shifts are induced by polar contacts with side chains of the peptide ligands. Hence, Aha is a versatile and minimally invasive vibrational label that is not only able to report on large structural changes during, e.g., protein folding, but also on very subtle changes of the electrostatic environment upon ligand binding.
非天然氨基酸叠氮基高丙氨酸（Aha）是一种环境敏感的红外探针，常用于特定位置的蛋白质结构和动力学研究。在此，通过将其整合到 PDZ2 结构域的结合槽附近，研究人员探索了该标签用于检测蛋白质-配体相互作用的能力。圆二色性和等温热滴定法测量结果表明，突变对蛋白质系统的干扰可以忽略不计，对蛋白质稳定性和结合亲和力的影响很小。二维红外光谱显示，当与两种不同的肽配体结合时，Aha 振动频率发生了小（1-3 cm）但可明显测量的红移，而伴随的分子动力学模拟表明，这些红移是由与肽配体侧链的极性接触引起的。因此，Aha 是一种通用且微创的振动标记，不仅能够报告蛋白质折叠等过程中的大结构变化，还能够报告配体结合时静电环境的微小变化。
