Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds.
Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds.
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DOI：
10.1016/s0021-9258(18)45990-x
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发表时间：
1992-01
期刊：
影响因子：
--
通讯作者：
K. Rothschild;Y. He;S. Sonar;T. Marti;H. Khorana
中科院分区：
文献类型：
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作者：
K. Rothschild;Y. He;S. Sonar;T. Marti;H. Khorana
利用定点突变体的傅里叶变换红外差光谱和时间分辨可见吸收光谱研究了Thr-46和Thr-89在细菌视紫红质光循环中的作用。Thr-46和Thr-89的取代揭示了相对于野生型细菌视紫红质，在光循环期间发色团和蛋白质结构的改变。与野生型相比，突变体T89 D和T89 A在较小程度上显示光适应状态的可见λ max的红移。在光循环期间，T89 A表现出增加的K中间体的衰减速率，而在室温下T89 D的光循环中未检测到K中间体。在T89 D的傅里叶变换红外差谱的羧基伸缩区域，早在K形成时就出现了一个新的谱带，这归因于Asp-89的去质子化。沿着该条带，在归属于Asp-212质子化的条带中出现强度增加。在突变体T46 V中，在L和M中间体中检测到Asp-96环境的扰动，这对应于其pK α的下降。这些数据表明，Thr-89位于靠近发色团，在全反式到13-顺式异构化过程中对其施加空间限制，并且可能参与延伸到Asp-212的氢键网络。此外，Thr-46和Asp-96之间的瞬时相互作用发生在光循环的早期。为了解释这些结果，以前提出的质子运输模型扩展到包括存在的氢键残基的瞬态网络。该模型可以解释细菌视紫红质光循环过程中关键氨基酸残基的质子化变化。
The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time-resolved visible absorption spectroscopy of site-directed mutants. Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin. The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type. During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature. In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89. Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212. In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha. These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212. In addition, a transient interaction between Thr-46 and Asp-96 occurs early in the photocycle. In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues. This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin.