Xie A, Kelemen L, Hendriks J, White B J, Hellingwerf K J, Hoff W D
Department of Physics, Oklahoma State University, 145 Physical Sciences II, Stillwater, OK 74078, USA.
Biochemistry. 2001 Feb 13;40(6):1510-7. doi: 10.1021/bi002449a.
Photoactive yellow protein (PYP) is a eubacterial photoreceptor and a structural prototype of the PAS domain superfamily of receptor and regulatory proteins. We investigate the activation mechanism of PYP using time-resolved Fourier transform infrared (FTIR) spectroscopy. Our data provide structural, kinetic, and energetic evidence that the putative signaling state of PYP is formed during a large-amplitude protein quake that is driven by the formation of a new buried charge, COO(-) of the conserved Glu46, in a highly hydrophobic pocket at the active site. A protein quake is a process consisting of global conformational changes that are triggered and driven by a local structural "fault". We show that large, global structural changes take place after Glu46 ionization via intramolecular proton transfer to the anionic p-coumarate chromophore, and are suppressed by the absence of COO(-) formation in the E46Q mutant. Our results demonstrate the significance of buried charge formation in photoreceptor activation. This mechanism may serve as one of the general themes in activation of a range of receptor proteins. In addition, we report the results of time-resolved FTIR spectroscopy of PYP crystals. The direct comparison of time-resolved FTIR spectroscopic data of PYP in aqueous solution and in crystals reveals that the structure of the putative signaling state is not developed in P6(3) crystals. Therefore, when the structural developments during the functional process of a protein are experimentally determined to be very different in crystals and solutions, one must be cautious in drawing conclusions regarding the functional mechanism of proteins based on time-resolved X-ray crystallography.
光活性黄色蛋白（PYP）是一种真细菌光感受器，也是受体和调节蛋白的PAS结构域超家族的结构原型。我们使用时间分辨傅里叶变换红外（FTIR）光谱研究PYP的激活机制。我们的数据提供了结构、动力学和能量方面的证据，表明PYP的假定信号状态是在一次大幅度的蛋白质震动过程中形成的，这次震动是由一个新的埋藏电荷——保守的Glu46的COO(-)——在活性位点高度疏水口袋中的形成所驱动的。蛋白质震动是一个由局部结构“缺陷”引发和驱动的全局构象变化过程。我们表明，在Glu46离子化后，通过分子内质子转移到阴离子对香豆酸发色团，会发生大规模的全局结构变化，并且在E46Q突变体中由于没有COO(-)形成而受到抑制。我们的结果证明了埋藏电荷形成在光感受器激活中的重要性。这种机制可能是一系列受体蛋白激活的一般主题之一。此外，我们报告了PYP晶体的时间分辨FTIR光谱结果。对PYP在水溶液和晶体中的时间分辨FTIR光谱数据的直接比较表明，假定信号状态的结构在P6(3)晶体中并未形成。因此，当通过实验确定蛋白质功能过程中的结构发展在晶体和溶液中有很大差异时，基于时间分辨X射线晶体学得出关于蛋白质功能机制的结论时必须谨慎。
