Schmidt Heather L Frericks, Shah Gautam J, Sperling Lindsay J, Rienstra Chad M
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
J Phys Chem Lett. 2010 May 4;1(10):1623-1628. doi: 10.1021/jz1004413.
Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pK(a) values of large, membrane or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble β1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly-(13)C,(15)N-labeled protein were monitored over several samples varying in pH; pK(a) values were determined from these shifts for E27, D36, and E42, and the bounds for the pK(a) of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pK(a) values give insights into the crystal packing of the protein.
带电残基在许多生物分子关键机制特征的定义中起着重要作用。用传统方法测定大型、膜蛋白或纤维状蛋白的pK(a)值可能具有挑战性。在本研究中，我们展示了如何利用固态核磁共振技术监测蛋白G的小可溶性β1免疫球蛋白结合结构域在pH滴定过程中的化学位移变化。在几个pH值不同的样品中，对整个均匀标记有(13)C、(15)N的蛋白中所有带电荷侧链氨基酸的化学位移进行了监测；从这些位移中确定了E27、D36和E42的pK(a)值，并确定了其他酸性侧链共振pK(a)的界限。此外，本研究还展示了计算得到的pK(a)值如何为蛋白质的晶体堆积提供见解。
