美国布兰代斯大学Dorothee Kern研究组在研究中取得进展。他们提出动态动态蛋白质集合中高能态的结构测定。该项研究成果发表在2022年3月2日出版的《自然》上。
他们开发了一种通过将 NMR 光谱衍生的伪接触位移(PCS) 与 Carr-Purcell-Meiboom-Gill (CPMG) 弛豫色散 6 (PCS-CPMG) 耦合来确定少数群体状态的高分辨率结构的方法。他们的方法还定义了高能偏移的相应动力学和热力学，从而表征了整个自由能景观。使用大量腺苷酸激酶 (Adk)、钙调蛋白和 Src 激酶的模拟数据，他们发现高能 PCS 可以准确地确定高能结构（均方根偏差小于 3.5 angström）。在催化过程中将他们的方法应用于 Adk，他们发现高能偏移涉及 AMP 和 ATP 盖的令人惊讶的小开口。这种以前未解决的高能结构解决了长期以来关于限制催化速率的构象相互转化的争议。
Adk 的高能结构为底物结合或产物释放做好了准备，表明将构象选择与该状态相结合的两步机制，然后是诱导拟合步骤进入完全闭合状态以催化磷酸转移反应。与其他解决高能状态的方法不同，如冷冻电镜和 X 射线晶体学，他们的解决方案 PCS-CPMG 方法在涉及较小系统（小于 60 kDa）和低至 0.5% 的群体的域重排的情况下表现出色，并能够在蛋白质发挥其功能的同时同时测定蛋白质结构、动力学和热力学。
附：英文原文
Title: Structure determination of high-energy states in a dynamic protein ensemble
Author: Stiller, John B., Otten, Renee, Hussinger, Daniel, Rieder, Pascal S., Theobald, Douglas L., Kern, Dorothee
Issue&Volume: 2022-03-02
Abstract: Macromolecular function frequently requires that proteins change conformation into high-energy states1,2,3,4. However, methods for solving the structures of these functionally essential, lowly populated states are lacking. Here we develop a method for high-resolution structure determination of minorly populated states by coupling NMR spectroscopy-derived pseudocontact shifts5 (PCSs) with Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion6 (PCS–CPMG). Our approach additionally defines the corresponding kinetics and thermodynamics of high-energy excursions, thereby characterizing the entire free-energy landscape. Using a large set of simulated data for adenylate kinase (Adk), calmodulin and Src kinase, we find that high-energy PCSs accurately determine high-energy structures (with a root mean squared deviation of less than 3.5angstrm). Applying our methodology to Adk during catalysis, we find that the high-energy excursion involves surprisingly small openings of the AMP and ATP lids. This previously unresolved high-energy structure solves a longstanding controversy about conformational interconversions that are rate-limiting for catalysis. Primed for either substrate binding or product release, the high-energy structure of Adk suggests a two-step mechanism combining conformational selection to this state, followed by an induced-fit step into a fully closed state for catalysis of the phosphoryl-transfer reaction. Unlike other methods for resolving high-energy states, such as cryo-electron microscopy and X-ray crystallography, our solution PCS–CPMG approach excels in cases involving domain rearrangements of smaller systems (less than 60kDa) and populations as low as 0.5%, and enables the simultaneous determination of protein structure, kinetics and thermodynamics while proteins perform their function.
DOI: 10.1038/s41586-022-04468-9
Source: https://www.nature.com/articles/s41586-022-04468-9
Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
官方网址：http://www.nature.com/
投稿链接：http://www.nature.com/authors/submit_manuscript.html
本期文章：《自然》：Online/在线发表
