Comparison of the Dielectric Response Obtained from Fluorescence Upconversion Measurements and Molecular Dynamics Simulations for Coumarin 153-Apomyoglobin Complexes and Structural Analysis of the Complexes by NMR and Fluorescence Methods
Comparison of the Dielectric Response Obtained from Fluorescence Upconversion Measurements and Molecular Dynamics Simulations for Coumarin 153-Apomyoglobin Complexes and Structural Analysis of the Complexes by NMR and Fluorescence Methods
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DOI：
10.1021/jp1008225
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发表时间：
2011-04-28
影响因子：
2.9
通讯作者：
Petrich, Jacob W.
中科院分区：
文献类型：
--
作者：
Bose, Sayantan;Adhikary, Ramkrishna;Petrich, Jacob W.
我们提出了一个比较的介电响应从荧光上转换实验和从分子动力学模拟的复合物的香豆素153与5 apoMbs：野生型马心（HH-WT）和野生型抹香鲸（SW-WT），其两个三重突变体，L29 F/H64 Q/V68 F和H64 L/V68 F/P88 A;和它的双突变体，L29 F/V68 L。野生型蛋白质的实验和模拟溶剂化弛豫函数C（t）s之间的比较范围从非常好到优秀。然而，对于我们研究的三种突变体，实验和模拟之间的一致性相当差。因此，进行了HH-WT复合物apoMb的复合物的NMR研究以及五种复合物的荧光能量转移和各向异性研究，以研究模拟所基于的结构。NMR测量证实了我们先前的结论，即C153位于HH-WT apoMb的血红素口袋中。对于野生型复合物，荧光能量转移测量提供了两个上升时间，表明两个尖端供体和C153受体之间存在明确的空间关系。这些结果证实了野生型复合物的结构完整性，并验证了用于分子动力学模拟的初始结构。另一方面，三种突变体提供了能量转移的单指数上升时间，表明模拟中使用的C153的位置可能是错误的，或者C153在能量转移实验的时间尺度上是移动的。荧光各向异性的研究也表明，双突变体是不完整的结构。此外，这些系统的检查证明了C153对其环境的敏感性，并允许观察血红素口袋中的差异。这些结果表明，在介电响应的研究中使用的改性蛋白质的结构表征的重要性，并建议进行分子动力学模拟的改性蛋白质的策略。
We present a comparison of the dielectric response obtained from fluorescence upconversion experiments and from molecular dynamics simulations of the complexes of coumarin 153 with five apomyoglobins (apoMbs): wild-type horse heart (HH-WT) and those of wild-type sperm whale (SW-WT); its two triple mutants, L29F/H64Q/V68F and H64L/V68F/P88A; and its double mutant, L29F/V68L. Comparisons between experimental and simulated solvation relaxation functions, C(t)s, for the wild-type proteins range from very good to excellent. For the three mutants we investigated, however, agreement between experiment and simulation was considerably inferior. Thus, an NMR study of the complex of the HH-WT complex apoMb, and fluorescence energy transfer and anisotropy studies of the five complexes, were performed to investigate the structures upon which the simulations were based. The NMR measurements confirm our earlier conclusions that the C153 lies in the heme pocket of the HH-WT apoMb. For the Wild-type complexes, fluorescence energy transfer measurements provide two rise times, suggesting g a definite spatial relationship between the two Tip donors and the C153 acceptor. These results confirm the structural integrity of the wild-type complexes and validate the initial structures used for the molecular dynamic; simulations. On the other hand, die three mutants provided single exponential rise times for energy transfer, suggesting that the position of the C153 used in the simulations may have been in error or that the C153 is mobile on the time, scale of the energy transfer experiment. Fluorescence anisotropy studies also suggest that the double mutant was not structurally intact. Furthermore, examination of these systems demonstrates the sensitivity of C153 to its environment and permits the observation of differences in the heme pockets. These results point to the importance of structural characterization of modified proteins used in studies of the dielectric response and suggest strategies for performing molecular dynamics simulations of modified proteins.