Probing the role of the heme distal and proximal environment in ligand dynamics in the signal transducer protein HemAT by time-resolved step-scan FTIR and resonance Raman spectroscopy
Probing the role of the heme distal and proximal environment in ligand dynamics in the signal transducer protein HemAT by time-resolved step-scan FTIR and resonance Raman spectroscopy
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通过时间分辨步进扫描 FTIR 和共振拉曼光谱探讨血红素远端和近端环境在信号转导蛋白 HemAT 配体动力学中的作用
DOI：
10.1021/acs.biochem.7b00558
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
and Eftychia Pinakoulaki
中科院分区：
文献类型：
--
作者：
Andrea Pavlou;Andreas Loullis;Hideaki Yoshimura;Shigetoshi Aono;and Eftychia Pinakoulaki
HemAT 是一种含有血红素的氧传感器蛋白，可控制趋气性。对分离的传感器结构域和全长 HemAT 蛋白以及 Y70F（B 螺旋）、L92A（E 螺旋）、T95A（E 螺旋）和 Y133F（G 螺旋）突变体进行时间分辨步进扫描 FTIR 研究，以阐明位点特异性突变对 CO 光解后配体动力学的影响。这些突变旨在扰乱血红素 Fe 结合气体配体 (CO) 和血红素近端环境附近的氢键和静电相互作用。 CO 与血红素 Fe 的重新结合在传感器结构域和全长 HemAT 以及突变体中是双相的，Y133F 突变体蛋白除外。 Y133F 中 CO 的单相重新结合表明，在 Y133 和血红素近端 H123 残基之间缺乏氢键的情况下，配体重新结合过程受到显着影响。近端环境的作用也通过共振拉曼光解离实验进行了探讨，其中传感器域 HemAT-CO 光产物的 Fe-His 模式在比差异共振拉曼光谱中脱氧形式更高的频率下被检测到。讨论了 Y133（G 螺旋）构象变化的作用以及远端 L92 和 T95 残基（E 螺旋）在调节血红素袋中配体动力学中的作用。
HemAT is a heme-containing oxygen sensor protein that controls aerotaxis. Time-resolved step-scan FTIR studies were performed on the isolated sensor domain and full-length HemAT proteins as well as on the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) mutants to elucidate the effect of the site-specific mutations on the ligand dynamics subsequent to CO photolysis. The mutations aimed to perturb H-bonding and electrostatic interactions near the heme Fe-bound gaseous ligand (CO) and the heme proximal environment. Rebinding of CO to the heme Fe is biphasic in the sensor domain and full-length HemAT as well as in the mutants, with the exception of the Y133F mutant protein. The monophasic rebinding of CO in Y133F suggests that in the absence of the H-bond between Y133 and the heme proximal H123 residue the ligand rebinding process is significantly affected. The role of the proximal environment is also probed by resonance Raman photodissociation experiments, in which the Fe–His mode of the photoproduct of sensor domain HemAT-CO is detected at a frequency higher than that of the deoxy form in the difference resonance Raman spectra. The role of the conformational changes of Y133 (G-helix) and the role of the distal L92 and T95 residues (E-helix) in regulating ligand dynamics in the heme pocket are discussed.