Diverse roles of glycine residues conserved in photoactive yellow proteins
Diverse roles of glycine residues conserved in photoactive yellow proteins
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DOI：
10.1529/biophysj.107.123414
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发表时间：
2008-05-01
影响因子：
3.4
通讯作者：
Kataoka, Mikio
中科院分区：
文献类型：
--
作者：
Imamoto, Yasushi;Tatsumi, Sanae;Kataoka, Mikio
以PERARNT SIM结构域蛋白的结构原型光活性黄蛋白为模板，通过丙氨酸扫描诱变研究了甘氨酸残基的作用。位于β -链末端的甘氨酸突变（Gly-37、Gly-59、Gly-86和Gly-115）导致了蛋白质结构的不稳定。另一方面，Gly-77和Gly-82的取代，加入到第5个a-螺旋中，使光循环减慢了15 - 20倍，这表明这些残基调节了光诱导结构在暗态结构和信号态结构之间的转换。最重要的是，大量的G29A处于漂白状态，并表现出1000倍慢的光循环。由于Glu-46的羧酸的O-epsilon 2与Gly-29的Ca的接触足够近，丙氨酸突变扰乱了这种包装。傅里叶变换红外光谱表明，gly -46的C=(epsilon 2)拉伸模式在G29A中上移了6 cm(-1)，表明Gly-29的C- α充当了与Glu-46形成的C- α - h中心点中心点O-epsilon 2氢键的质子供体，稳定了暗态结构。在光循环过程中，通过向发色团提供一个质子，Glu-46带负电荷，导致疏水填料的破裂，从而导致蛋白质的构象变化。
The role of glycine residues was studied by alanine-scanning mutagenesis using photoactive yellow protein, a structural prototype of PERARNT SIM domain proteins, as a template. Mutation of glycine located close to the end of beta-strands with dihedral angles disallowed for alanine (Gly-37, Gly-59, Gly-86, and Gly-115) induces destabilization of the protein structure. On the other hand, substitution for Gly-77 and Gly-82, incorporated into the fifth a-helix, slows the photocycle by 15 - 20 times, suggesting that these residues regulate the light-induced structural switch between dark-state structure and signaling-state structure. Most importantly, a significant amount of G29A is in the bleached state and showed a 1000-fold slower photocycle. As O-epsilon 2 of the carboxylic acid of Glu-46 is close enough for contact with Ca of Gly-29, alanine mutation perturbs this packing. Fourier transform infrared spectroscopy demonstrated that the C=(epsilon 2) stretching mode of Glu-46 is 6 cm (-1) upshifted in G29A, suggesting that C-alpha of Gly-29 acts as a proton donor for the C-alpha-H center dot center dot center dot O-epsilon 2 hydrogen bond with Glu-46, which stabilizes the dark-state structure. During the photocycle, Glu-46 becomes negatively charged by donating a proton to the chromophore, resulting in breakage of this hydrophobic packing and consequent conformational change of the protein.