Comprehensive Determination of Protein Tyrosine pKa Values for Photoactive Yellow Protein Using Indirect 13C NMR Spectroscopy
Comprehensive Determination of Protein Tyrosine pKa Values for Photoactive Yellow Protein Using Indirect 13C NMR Spectroscopy
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DOI：
10.1016/j.bpj.2011.12.024
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发表时间：
2012-02-08
影响因子：
3.4
通讯作者：
Mulder, Frans A. A.
中科院分区：
文献类型：
--
作者：
Oktaviani, Nur Alia;Pool, Trijntje J.;Mulder, Frans A. A.
在蓝光照射下，嗜盐盐螺菌能够调节其鞭毛马达的活动，从而逃避潜在有害的紫外线辐射。 14 kDa 可溶性胞浆光敏黄色蛋白 (PYP) 被认为是这种避光反应的主要介质，并产生与细菌运动光谱密切匹配的紫外/可见吸收光谱。在电子基态下，PYP 的对香豆酸 (pCA) 发色团带负电，并与 Glu-46 和 Tyr-42 的侧链形成两个短氢键。由此产生的酸三联体是 PYP 光学吸收弛豫动力学显着 pH 依赖性的核心。在这里，我们描述了一种 NMR 方法，用于序列特异性地跟踪 PYP 从 pH 3.41 到 11.24 的所有酪氨酸侧链质子化状态。非质子化C-13(gamma)的间接观察。敏感且分辨率良好的二维 C-13-H-1 光谱中的共振被证明是这项工作的关键，因为对其他环系统共振的观察受到光谱拥塞和环翻转导致的谱线展宽的阻碍。我们观察到 PYP 中的三类酪氨酸残基表现出非常不同的 pK(a) 值，具体取决于酚侧链是否暴露于溶剂、埋藏或氢键结合。特别是，我们的数据表明，Tyr-42 在 3.41-11.24 的 pH 范围内保持完全质子化，并且在 PYP 光循环动力学中观察到的 pH 引起的变化不能由 Tyr-42 电荷态的变化引起。因此，pCA 发色团在电子基态下的静电相互作用不太可能发生变化。
Upon blue-light irradiation, the bacterium Halorhodospira halophila is able to modulate the activity of its flagellar motor and thereby evade potentially harmful UV radiation. The 14 kDa soluble cytosolic photoactive yellow protein (PYP) is believed to be the primary mediator of this photophobic response, and yields a UV/Vis absorption spectrum that closely matches the bacterium's motility spectrum. In the electronic ground state, the para-coumaric acid (pCA) chromophore of PYP is negatively charged and forms two short hydrogen bonds to the side chains of Glu-46 and Tyr-42. The resulting acid triad is central to the marked pH dependence of the optical-absorption relaxation kinetics of PYP. Here, we describe an NMR approach to sequence-specifically follow all tyrosine side-chain protonation states in PYP from pH 3.41 to 11.24. The indirect observation of the nonprotonated C-13(gamma). resonances in sensitive and well-resolved two-dimensional C-13-H-1 spectra proved to be pivotal in this effort, as observation of other ring-system resonances was hampered by spectral congestion and line-broadening due to ring flips. We observe three classes of tyrosine residues in PYP that exhibit very different pK(a) values depending on whether the phenolic side chain is solvent-exposed, buried, or hydrogen-bonded. In particular, our data show that Tyr-42 remains fully protonated in the pH range of 3.41-11.24, and that pH-induced changes observed in the photocycle kinetics of PYP cannot be caused by changes in the charge state of Tyr-42. It is therefore very unlikely that the pCA chromophore undergoes changes in its electrostatic interactions in the electronic ground state.