Blue light-induced dimerization of a bacterial LOV-HTH DNA-binding protein.
Blue light-induced dimerization of a bacterial LOV-HTH DNA-binding protein.
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DOI：
10.1021/bi401040m
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发表时间：
2013-09-24
期刊：
影响因子：
2.9
通讯作者：
Gardner, Kevin H.
中科院分区：
文献类型：
--
作者：
Zoltowski, Brian D.;Motta-Mena, Laura B.;Gardner, Kevin H.
光敏蛋白质利用光驱动的变构来控制生物化学活动，作为基础科学和工程界使用的模型系统和新型试剂引起了极大的兴趣。一种这样的蛋白质，来自海洋细菌Erythrobacter litoralis HTCC 2594的光激活EL 222转录因子，对此类研究很有吸引力，因为它利用蓝光驱动光-氧-电压（LOV）感觉和螺旋-转向-阻碍（HTH）效应结构域的重新定位，以允许在自然和人工系统中光激活基因转录。这一过程所需的蛋白质构象变化尚未得到很好的理解，部分原因是EL 222光激发态的寿命相对较短（τ~29 s），这使得某些生物物理方法的表征复杂化。在这里，我们报告了我们如何通过创建具有V41 I，L52 I，A79 Q和V121 I点突变（AQTrip）的EL 222变体来规避这种限制，该变体稳定了光活化状态。使用野生型和AQTrip EL 222蛋白，我们结合溶液散射、核磁共振和电迁移率变化测定来探测EL 222的激活。尺寸排阻色谱和光散射表明，AQTrip寡聚化在DNA的情况下，并选择在DNA底物的存在下的EL 222-二聚体-DNA复合物。这些结果在具有稳定复合物的高亲和力DNA结合位点的野生型EL 222中得到证实。EL 222-DNA复合物的NMR分析证实了在先前表征的DNA底物存在下的2：1化学计量。结合起来，这些新的方法已经验证了一个关键的机制步骤，即蓝光通过LOV和HTH界面诱导EL 222二聚化。
With their utilization of light-driven allostery to control biochemical activities, photosensory proteins are of great interest as model systems and novel reagents for use by the basic science and engineering communities. One such protein, the light-activated EL222 transcription factor, from the marine bacterium Erythrobacter litoralis HTCC2594, is appealing for such studies, as it harnesses blue light to drive the reorientation of Light-Oxygen-Voltage (LOV) sensory and Helix-Turn-Helix (HTH) effector domains to allow photoactivation of gene transcription in natural and artificial systems. The protein conformational changes required for this process are not well understood, due in part to the relatively short lifetime of the EL222 photoexcited state (τ~29 s) which complicates its characterization with certain biophysical methods. Here we report how we have circumvented this limitation by creating an EL222 variant harboring V41I, L52I, A79Q and V121I point mutations (AQTrip) that stabilizes the photoactivated state. Using the wild-type and AQTrip EL222 proteins, we have probed EL222 activation using a combination of solution scattering, NMR and electromobility shift assays. Size exclusion chromatography and light scattering indicates that AQTrip oligomerizes in the absence of DNA, and selects for an EL222-dimer-DNA complex in the presence of DNA substrates. These results are confirmed in wild-type EL222 with a high-affinity DNA binding site that stabilizes the complex. NMR analyses of the EL222-DNA complex confirm a 2:1 stoichiometry in the presence of a previously characterized DNA substrate. Combined, these novel approaches have validated a key mechanistic step, whereby blue-light induces EL222 dimerization through LOV and HTH interfaces.
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影响因子：
3.6
作者：
Pinto UM;Winans SC
通讯作者：
Winans SC
影响因子：
2.9
作者：
Halavaty, Andrei S.;Moffat, Keith
通讯作者：
Moffat, Keith
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A
DOI：
10.1038/nsb845
发表时间：
2002-10-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Maris, AE;Sawaya, MR;Dickerson, RE
通讯作者：
Dickerson, RE
影响因子：
5.6
作者：
Moeglich, Andreas;Moffat, Keith
通讯作者：
Moffat, Keith