Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization-enhanced solid-state NMR
Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization-enhanced solid-state NMR
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DOI：
10.1073/pnas.0900908106
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发表时间：
2009-06-09
影响因子：
11.1
通讯作者：
Griffin, Robert G.
中科院分区：
文献类型：
--
作者：
Bajaj, Vikram S.;Mak-Jurkauskas, Melody L.;Griffin, Robert G.
蛋白质反应中间体的观察和结构研究具有挑战性，因为状态的混合物通常以低浓度存在。在这里，我们使用一个250 GHz的回旋（回旋共振脉泽）和低温温度进行高频动态核极化（DNP）NMR实验，提高灵敏度的魔角旋转NMR光谱的低温捕获的光循环中间体的细菌视紫红质（bR）的一个因素，约90。多维光谱的U-C-13，N-15-标记的样品解决共存状态，并允许化学位移分配在亚视黄基发色团的几个中间体以前没有观察到。的相关光谱揭示了意想不到的异质性在黑暗中适应的bR，在K状态的失真，最重要的是，4个离散的L子状态。L的混合物的热弛豫表明，这些子状态中的3个回复到bR（568），并且只有具有最强的抗衡和完全弛豫的13-顺式键的1个子状态是功能性的。这些明确的观察功能和分流状态的bR光循环提供了一个预览的机制的见解，将通过灵敏度增强的DNP NMR膜蛋白。如果没有来自DNP的信号增强，这些观察是不可能的。
Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron ( cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of approximate to 90. Multidimensional spectroscopy of U-C-13, N-15-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR(568) and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP.