Allosteric transitions in cytochrome P450eryF explored with pressure-perturbation spectroscopy, lifetime FRET, and a novel fluorescent substrate, Fluorol-7GA.
Allosteric transitions in cytochrome P450eryF explored with pressure-perturbation spectroscopy, lifetime FRET, and a novel fluorescent substrate, Fluorol-7GA.
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DOI：
10.1021/bi8011803
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发表时间：
2008-10-28
期刊：
影响因子：
2.9
通讯作者：
Halpert, James R.
中科院分区：
文献类型：
--
作者：
Davydov, Dmitri R.;Davydova, Nadezhda Y.;Halpert, James R.
为了建立一种在高静水压力下监测细胞色素P450eryF中底物结合的直接方法，我们引入了一种激光染料氟- 7ga （F7GA）作为一种新型荧光配体。P450的高荧光强度和合理的吸收带激发带分辨率使我们能够建立与压力扰动兼容的高灵敏度结合试验。F7GA与P450eryF的相互作用导致了充分的自旋位移，揭示了协同结合（S50 = 8.2±1.3µM, n = 2.3±0.1）。荧光共振能量转移（FRET）实验表明，存在至少两个底物结合位点，表观KD值在0.1 - 0.3和6 - 9µM范围内。与CYP3A4的早期观察类似，增加静水压力不会导致底物配合物的完全解离或自旋平衡向低自旋状态的位移。相反，增加的压力增强了f7ga诱导的自旋位移的协同性，因此Hill系数在2 kbar时接近3。终生FRET实验显示，在高压下，酶对F7GA的亲和力显著增加，这表明配体的结合诱导了与蛋白质水合作用重要增加相关的构象转变。这种转变在很大程度上削弱了血红素口袋的溶剂可及性，并导致高自旋底物结合酶在高压下具有不寻常的稳定性。
To establish a direct method to monitor substrate binding in cytochrome P450eryF applicable at elevated hydrostatic pressures we introduce a laser dye Fluorol-7GA (F7GA) as a novel fluorescent ligand. The high intensity of fluorescence and reasonable resolution of the excitation band from the absorbance bands of P450 allowed us to establish highly sensitive binding assays compatible with pressure perturbation. The interactions of F7GA with P450eryF cause an ample spin shift revealing cooperative binding (S50 = 8.2 ± 1.3 µM, n = 2.3 ± 0.1). Fluorescence resonance energy transfer (FRET) experiments suggest the presence of at least two substrate binding sites with apparent KD values in the ranges of 0.1 – 0.3 and 6 – 9 µM. Similar to earlier observations with CYP3A4, increasing hydrostatic pressure does not cause either a complete dissociation of the substrate complexes or a displacement of the spin equilibrium towards the low-spin state. Rather, increased pressure enhances the cooperativity of the F7GA-induced spin shift, so that the Hill coefficient approaches 3 at 2 kbar. Lifetime FRET experiments revealed an important increase in the affinity of the enzyme for F7GA at elevated pressures, suggesting that the binding of the ligand induces a conformational transition associated with an important increase in protein hydration. This transition largely attenuates the solvent accessibility of the heme pocket and causes an unusual stability of the high-spin, substrate-bound enzyme at elevated pressures.
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影响因子：
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作者：
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通讯作者：
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DOI：
10.1016/j.bbrc.2003.09.247
发表时间：
2003-12-05
影响因子：
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发表时间：
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DOI：
10.1073/pnas.050406897
发表时间：
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影响因子：
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