The Fritz Haber Research Center, Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
J Am Chem Soc. 2010 Aug 18;132(32):11093-102. doi: 10.1021/ja1010652.
"Proton-collecting antenna" are conjectured to consist of several carboxylates within hydrogen-bond (HB) networks on the surface of proteins, which funnel protons to the orifice of an internal proton wire leading to the protein's active site. Yet such constructions were never directly visualized. Here we report an X-ray structure of green fluorescent protein (GFP) of the highest resolution to date (0.9 A). It allows the identification of some pivotal hydrogen atoms pertinent to uncertainties concerning the protonation state of the chromophore. Applying a computer algorithm for mapping proton wires in proteins reveals the previously observed "active site wire" connecting Glu222 with the surface carboxylate Glu5. In addition, it is now possible to identify what appears to be a proton-collecting apparatus of GFP. It consists of a negative surface patch containing carboxylates, threonines, and water molecules, connected by a HB network to Glu5. Furthermore, we detect exit points via Asn146 and His148 to a hydrophobic surface region. The more extensive HB network of the present structure, as compared with earlier GFP structures, is not accidental. A systematic investigation of over 100 mutants shows a clear correlation between the observed water content of GFP X-ray structures and their resolution. With increasing water content, the proton wires become progressively larger. These findings corroborate the scenario in which the photodissociated proton from wild-type GFP can leak outside, whereafter another proton is recruited via the proton-collecting apparatus reported herein.
“质子收集天线”被推测由蛋白质表面氢键 (HB) 网络中的几个羧酸盐组成，这些羧酸盐将质子引导到通向蛋白质活性位点的内部质子导线的孔口。然而，这些结构从未被直接观察到。在这里，我们报告了迄今为止分辨率最高的绿色荧光蛋白 (GFP) 的 X 射线结构（0.9Å）。它允许识别一些与发色团质子化状态有关的关键氢原子。应用一种用于在蛋白质中映射质子导线的计算机算法，揭示了先前观察到的将 Glu222 与表面羧酸盐 Glu5 连接起来的“活性位点导线”。此外，现在可以识别 GFP 的质子收集装置。它由一个含有羧酸盐、苏氨酸和水分子的负表面斑块组成，通过 HB 网络与 Glu5 相连。此外，我们通过 Asn146 和 His148 检测到通往疏水表面区域的出口点。与早期 GFP 结构相比，当前结构更广泛的 HB 网络并非偶然。对 100 多个突变体的系统研究表明，GFP X 射线结构的观察到的含水量与其分辨率之间存在明显的相关性。随着含水量的增加，质子导线变得越来越大。这些发现证实了野生型 GFP 光解分离的质子可以泄漏到外部，随后通过本文报道的质子收集装置招募另一个质子的情景。
