Phytochrome photochromism probed by site-directed mutations and chromophore esterification
Phytochrome photochromism probed by site-directed mutations and chromophore esterification
复制标题
DOI：
10.1021/ja972875s
复制
发表时间：
1997-12-03
影响因子：
15
通讯作者：
Song, PS
中科院分区：
文献类型：
--
作者：
Bhoo, SH;Hirano, T;Song, PS
光敏色素是一种重要的光致变色蛋白；它们作为光传感器和/或开关，用于红光/远红光诱导植物的形态发生和发育反应，如萌发、开花和基因表达；有关综述，请参阅参考文献1-3。光致变色剂的光致变色性表现为红光吸收Pr形式(λmax 666 nm)和远红光吸收PfR形式(λmax 730 nm)之间的可逆异构化。4-6游离生色团能够在溶液中发生光致异构化，7，8，但不表现出光致变色蛋白的光致可逆光谱移位特性。9在1124个氨基酸残基中确定赋予光敏色素光致变色性的关键氨基酸残基是一项具有挑战性的任务。我们的策略始于系统地截断光敏色素A的N端和C端(全光敏色素A的光敏色素A，光敏色素A的光敏色素A，以及光敏色素A基因的光敏A)，并在发色团附近进行定点突变(SDM)。10，11我们现在可以将SDM定位于与PhyA光致变色有关的特定氨基酸残基。我们还探讨了丙酸侧链电荷对光致变色的作用。在这项研究中，我们使用藻蓝胆素(PCB)进行全蛋白的共价重组[以下简称PhyA，除非另有说明]，如前所述。9-11通过替换发色团半胱氨酸-323附近的5个保守氨基酸残基(D309、R318、H321、H324和Q326)而产生的10个突变体中，只有H324残基被鉴定为对PhyA的光致变色特性至关重要。突变株H324R和H324L在较高温度下通过锌离子印迹检测到发色团连接水平，但没有检测到光致变色。11突变体H324G再次证实了H324对光致变色的关键重要性(表1)。突变体H324F能自动催化连接PCB发色团，但不显示光致变色性。而突变株H324Q有效地组装了发色团，并表现出特征的光致变色性。这种功能突变的保留/获得并不令人惊讶，因为谷氨酰胺残基具有立体要求和类似组氨酸的氢键基团，经常可以取代野生型蛋白中的后一种残基以保留或增强活性。13位于Cys-323发色团附近的Y327F、S322V和P320M等其他高度保守的氨基酸残基的替代突变在其自催化裂解酶活性光致变色行为中是正常的。我们之前的研究也表明，除了His-324以外，发色团附近的其他氨基酸残基也起到了相当被动的作用。11最近提出了以丝氨酸-322为固定催化残基的光敏色素光致变色的光互变机理。14我们使用表1中的S322V突变体的结果表明，该残基在PhyA的光致变色中不起关键作用。从N端截断到残基80，消除了水稻Phya的光致变色性。15在本研究中，从N端到残基74和78的缺失突变体都表现出正常的裂解酶活性和光致变色性。然而，残基81的缺失取消了连接和光致变色性(表1)。N端74缺失突变体中的电荷改变K78E突变和疏水性Q81L突变保留了光致变色特性(表1)。然而，在N-末端-81片段中，异亮氨酸-80被证明是关键残基，描绘了参与光致变色的多肽片段的临界边界。…
Phytochromes are remarkable photochromic proteins; they serve as light sensors and/or switches for red/far-red lightinduced morphogenic and developmental responses such as germination, flowering, and gene expression in plants; for review, see refs 1-3. The photochromism of phytochromes is manifested by the photoreversible isomerization between the red light absorbing Pr form (λmax 666 nm) and the far-red light absorbing Pfr form (λmax 730 nm). 4-6 The free chromophore is capable of photoisomerization in solution, 7, 8 but does not exhibit photoreversible spectral shifts characteristic for the photochromic proteins. 9 To identify the critical amino acid residues, among 1124 residues, that confer photochromism to phytochromes is a challenging task. Our strategy began with systematic N-and C-terminal truncations of phytochrome A (phyA for holophytochrome A, PHYA for apophytochrome A, and PHYA for PHYA gene) and site-directed mutagenesis (SDM) in the vicinity of the chromophore site. 10, 11 We are now in a position to target SDM at specific amino acid residues involved in the photochromism of phyA. We also probed the role of the propionate side chain charges on the photochromism. For this study, we used phycocyanobilin (PCB) for covalent reconstitution of holoprotein [hereafter, phyA unless otherwise specified], as described previously. 9-11 Of the ten mutants generated by substituting the five conserved amino acid residues (D309, R318, H321, H324, and Q326) in the vicinity of the chromophore-cysteine-323, only the H324 residue was identified as being critical for the photochromic properties of phyA. Mutants H324R and H324L showed a detectable level of chromophore ligation by Zn2+-blot at a higher temperature, but without detectable photochromism. 11 The critical importance of H324 for photochromism is reconfirmed with mutant H324G (Table 1). Mutant H324F autocatalytically ligated the PCB chromophore, but failed to display photochromism. However, mutant H324Q assembled the chromophore efficiently and exhibited the characteristic photochromism. This “retention/gain of function mutation” is not surprising since glutamine residue with its steric requirement and H-bonding groups similar to that of histidine can often substitute the latter residue in wild type proteins with retention or enhancement of activity. 13Substitution mutants of other highly conserved amino acid residues including Y327F, S322V, and P320M in the immediate vicinity of the Cys-323 chromophore site were normal in their autocatalytic lyase activity photochromic behaviors. Our previous study also showed rather passive roles of other amino acid residues within the chromophore vicinity, except for His-324. 11 The phototautomeric mechanism for the phytochrome photochromism on the basis of serine-322 as an anchimeric catalytic residue has been proposed recently. 14 Our results using S322V mutant in Table 1 indicate that this residue does not play a critical role for photochromism in phyA. Truncation from the N-terminus to residue 80 abolished the photochromism of rice phyA. 15 In the present study, deletion mutants from the N-terminus to residues 74 and 78 both exhibited normal lyase activity and photochromism. However, deletion to residue 81 abolished the ligation and photochromism (Table 1). The charge-altering K78E mutation and the hydrophobicity-imposing Q81L mutation within the N-terminus-74 deletion mutant retained the photochromic properties (Table 1). However, within the N-terminus-81 segment, isoleucine-80 turned out to be the critical residue, delineating the critical boundary for the peptide segment involved in photochromism …