Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
J Phys Chem B. 2013 Oct 24;117(42):13082-9. doi: 10.1021/jp402772x. Epub 2013 Jul 9.
Electrostatic and conformational heterogeneity make central contributions to protein function, but their experimental characterization requires a combination of spatial and temporal resolution that is challenging to achieve. Src homology 3 (SH3) domains mediate protein-protein interactions, and NMR studies have demonstrated that most possess conformational heterogeneity, which could be critical for their function. Here, we use the IR absorptions of carbon-deuterium (C-D) bonds site-selectively incorporated throughout the N-terminal SH3 domain from the murine adapter protein Crk-II to characterize its different microenvironments with high spatial and temporal resolution. The C-D absorptions are only differentiated in the folded state of the protein where they show evidence of significant environmental heterogeneity. However, the spectra of the folded state are independent of temperature, and upon thermal denaturation the protein undergoes a single, global unfolding transition. While some evidence of conformational heterogeneity is found within the peptide backbone, the majority of the environmental heterogeneity appears to result from electrostatics.
静电和构象异质性对蛋白质功能有重要贡献，但要对其进行实验表征，需要结合空间和时间分辨率，这是极具挑战性的。Src 同源 3（SH3）结构域介导蛋白质-蛋白质相互作用，NMR 研究表明，大多数 SH3 结构域都具有构象异质性，这对其功能可能至关重要。在这里，我们使用从鼠源衔接蛋白 Crk-II 的 N 端 SH3 结构域中掺入的全碳-氘（C-D）键的红外吸收，以高空间和时间分辨率来表征其不同的微环境。C-D 吸收仅在蛋白质的折叠状态下有所区分，在该状态下，它们显示出明显的环境异质性的证据。然而，折叠状态的光谱不随温度而变化，并且在热变性时，蛋白质经历单一的、全局展开转变。虽然在肽骨架内发现了一些构象异质性的证据，但大部分环境异质性似乎是由静电作用引起的。
