Blue‐Light Induced Interaction of LOV Domains from Chlamydomonas reinhardtii
Blue‐Light Induced Interaction of LOV Domains from Chlamydomonas reinhardtii
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蓝光诱导莱茵衣藻 LOV 结构域的相互作用
DOI：
10.1002/cbic.200800158
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发表时间：
1938
期刊：
影响因子：
3.2
通讯作者：
B. Dick
中科院分区：
文献类型：
--
作者：
R. J. Kutta;E. S. A. Hofinger;H. Preuss;G. Bernhardt;B. Dick
莱茵衣藻的趋光素是一种分子量为120 kDa的蓝光受体，在这种绿色衣藻的配子发生中起关键作用。它包含两个称为LOV 1和LOV 2（光氧和电压）的光敏结构域和一个丝氨酸/苏氨酸激酶结构域。后结合的发色团是黄素单胞苷肽（FMN）。在吸收蓝光后，LOV结构域经历激活Ser/Thr激酶的光循环。这种激活的机制仍然是未知的。我们研究了重组C.通过UV/维斯光谱法、尺寸排阻色谱法（SEC）和戊二醛化学交联法测定了其分子量。在强蓝光脉冲后通过UV/维斯光谱监测的LOV 1从信号状态到暗状态的热逆反应不能用单指数模型解释，尽管光谱没有表明存在额外的物质。因此，我们在黑暗中通过尺寸排阻色谱法研究了LOV 1结构域的四级结构。这揭示了在天然条件下二聚体和更高级寡聚体（MW>200 kDa）之间的平衡。通过SEC未检测到单体。然而，通过蛋白质与戊二醛交联的平衡分析和随后的SDS-PAGE，鉴定了单体和二聚体。LOV 1暴露于蓝光导致单体/二聚体比率降低，然后在黑暗中重新平衡。晶体结构中存在的LOV 1二聚体的溶剂可及表面积和Conolly表面的计算支持实验观察结果，即在天然状态下未检测到单体。提出了一个模型，该模型解释了蓝光驱动的LOV 1结构域四级结构的变化，并暗示了含LOV蛋白质中光激活和调节的分子基础。
The phototropin fromChlamydomonas reinhardtiiis a 120 kDa blue light receptor that plays a key role in gametogenesis of this green alga. It comprises two light‐sensing domains termed LOV1 and LOV2 (lightoxygen andvoltage) and a serine/threonine kinase domain. The post‐translationally incorporated chromophore is flavin mononucleotide (FMN). Upon absorption of blue light, LOV domains undergo a photocycle that activates a Ser/Thr kinase. The mechanism of this activation is still unknown. We studied the oligomerization of the recombinant LOV1 domain (amino acids 16–133) ofC. reinhardtiiby means of UV/Vis spectroscopy, size‐exclusion chromatography (SEC), and chemical cross‐linking with glutardialdehyde. The thermal back‐reaction of LOV1 from the signaling state to the dark state as monitored by UV/Vis spectroscopy after an intensive blue light pulse could not be explained by a monoexponential model, although the spectra did not indicate the presence of an additional species. Therefore, we investigated the quaternary structure of the LOV1 domain by size‐exclusion chromatography in the dark. This revealed an equilibrium between dimers and higher oligomers (MW>200 kDa) under native conditions. No monomers were detected by SEC. However, by analysis of the equilibrium by cross‐linking of the protein with glutardialdehyde and subsequent SDS‐PAGE, monomers and dimers were identified. Exposure of LOV1 to blue light resulted in a decrease in the monomer/dimer ratio, followed by re‐equilibration in the dark. Calculation of the solvent‐accessible surface area and the Conolly surfaces of the LOV1 dimers present in the crystal structure support the experimental observation that no mononomers are detected in the native state. A model is presented that accounts for a blue‐light‐driven change in the quaternary structure of the LOV1 domain and gives hints to the molecular basis of light activation and regulation in LOV‐containing proteins.
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通过戊二醛交联研究猪心乳酸脱氢酶的重新结合
DOI：
10.1515/znc-1981-9-1013
发表时间：
1981
期刊：
Zeitschrift für Naturforschung C
影响因子：
--
作者：
G. Bernhardt;R. Rudolph;R. Jaenicke
通讯作者：
R. Jaenicke
影响因子：
2.9
作者：
Crosson, S;Rajagopal, S;Moffat, K
通讯作者：
Moffat, K
影响因子：
4.3
作者：
Knieb, E;Salomon, M;Rüdiger, W
通讯作者：
Rüdiger, W
影响因子：
7.4
作者：
PALMER, JM;SHORT, TW;BRIGGS, WR
通讯作者：
BRIGGS, WR
影响因子：
3.1
作者：
Buttani, Valentina;Losi, Aba;Gaertner, Wolfgang
通讯作者：
Gaertner, Wolfgang