Active site mutants implicate key residues for control of color and light cycle kinetics of photoactive yellow protein
Active site mutants implicate key residues for control of color and light cycle kinetics of photoactive yellow protein
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DOI：
10.1021/bi9622884
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发表时间：
1997-01-07
期刊：
影响因子：
2.9
通讯作者：
Getzoff, ED
中科院分区：
文献类型：
--
作者：
Genick, UK;Devanathan, S;Getzoff, ED
为了了解光敏蛋白质的蛋白质和发色团成分如何相互作用来创建光循环，我们对光活性黄色蛋白(PYP)的定点突变体进行了时间分辨光谱，最近确定了PYP在基态和无色I2态的晶体结构，使我们能够设计突变体并研究它们在原子级的感光性质。我们建立了PYP辅基蛋白的快速诱变和异源细菌表达系统，并通过与天然蛋白中的对羟基肉桂酸发色团形成共价硫酯键来产生全蛋白。天然蛋白中发现Glu46被Gin取代，并在基态与发色团的酚氧发生氢键作用，Glu46Gln I章动使基态吸收峰从446 Co462 nm移动到446 Co462 nm，这表明PYP的颜色可以通过氢键的变化来微调，Ala，Ala取代了基态活性中心和溶剂之间的Arg52Arg52Ala突变体的较小珊瑚礁位移(至452 nm)表明，与Arg52的静电相互作用对生色团上的电荷稳定并不重要。这两种突变都会导致光循环动力学发生有趣的变化。最显著的影响是Glu46Gln PYP的基态恢复速率增加了700倍。在pH从5到10(pK(D)=8)的响应下，我们仔细地重新考察了pH对野生型PYP光循环的影响，随着pH从5增加到10，褪色速率下降了约3倍。有色基态的恢复速率呈现钟形的pH依赖关系，受两个pk(A)值(6.4和9.4)控制。在pH值为7.9时，Arg52Ala的最大回收率约为pH值为5时的16倍。pH对Arg52Ala的影响与野生型相似，只是褪色快，恢复慢。这些突变的动力学效应和随pH的变化表明，PYP光周期的两个阶段都受到蛋白质成分的积极控制。
TO understand how the protein and chromophore components of a light-sensing protein interact to create a light cycle, we performed time-resolved spectroscopy on site-directed mutants of photoactive yellow protein (PYP), Recently determined crystallographic structures of PYP in the ground and colorless I2 states allowed us to design mutants and to study their photosensing properties at the atomic revel. We developed a system for rapid mutagenesis and heterologous bacterial expression for PYP apoprotein and generated holoprotein through formation of a covalent thioester linkage with the p-hydroxycinnamic acid chromophore as found in the native protein, Glu46, replaced by Gin, is buried in the active site and hydrogen bonds to the chromophore's phenolate oxygen in the ground state, The Glu46Gln I-nutation shifted the ground state absorption maximum from 446 Co 462 nm, indicating that the color of PYP can be fine-tuned by the alteration of hydrogen bonds, Arg52, which separates the active site from solvent in the ground state, was substituted by Ala, The smaller reef shift (to 452 nm) of the Arg52Ala mutant suggests that electrostatic interactions with Arg52 are not important for charge stabilization on the chromophore. Both mutations cause interesting changes in light cycle kinetics. The most dramatic effect is a 700-fold increase in the rate of recovery to the ground state of Glu46Gln PYP in response to a change in pH from pH 5 to 10 (pK(d) = 8), Prompted by this large effect, we conducted a careful reexamination of pH effects on the wild-type PYP light cycle, The rate of color loss decreased about 3-fold with increasing pH from pH 5 to 10. The rate of recovery to the colored ground state showed a bell-shaped pH dependence, controlled by two pK(a) values (6.4 and 9.4). The maximum recovery rate at pH 7.9 is about 16 times faster than at pH 5. The effect of pH on Arg52Ala is like that on wild type except for faster loss of color and slower recovery. These kinetic effects of the mutations and the changes with pH demonstrate that both phases in PYP's light cycle are actively controlled by the protein component.