Timerghazin Qadir K, Carlson Haley J, Liang Chen, Campbell Robert E, Brown Alex
University of Alberta, Department of Chemistry, Edmonton, Alberta T6G 2G2, Canada.
J Phys Chem B. 2008 Feb 28;112(8):2533-41. doi: 10.1021/jp709900k. Epub 2008 Feb 5.
By virtue of its self-sufficiency to form a visible wavelength chromophore within the confines of its tertiary structure, the Aequorea victoria green fluorescent protein (GFP) is single-handedly responsible for the ever-growing popularity of fluorescence imaging of recombinant fusion proteins in biological research. Engineered variants of GFP with altered excitation or emission wavelength maxima have helped to expand the range of applications of GFP. The engineering of the GFP variants is usually done empirically by genetic modifications of the chromophore structure and/or its environment in order to find variants with new photophysical properties. The process of identifying improved variants could be greatly facilitated if augmented or guided by computational studies of the chromophore ground and excited-state properties and dynamics. In pursuit of this goal, we now report a thorough investigation of computational methods for prediction of the absorbance maxima for an experimentally validated series of engineered GFP chromophore analogues. The experimental dataset is composed of absorption maxima for 10 chemically distinct GFP chromophore analogues, including a previously unreported Y66D variant, measured under identical denaturing conditions. For each chromophore analogue, excitation energies and oscillator strengths were calculated using configuration interaction with single excitations (CIS), CIS with perturbative correction for double substitutions [CIS(D)], and time-dependent density functional theory (TD DFT) using several density functionals with solvent effects included using a polarizable continuum model. Comparison of the experimental and computational results show generally poor quantitative agreement with all methods attempted. However, good linear correlations between the calculated and experimental excitation energies (R2>0.9) could be obtained. Oscillator strengths obtained with TD DFT using pure density functionals also correlate well with the experimental values. Interestingly, most of the computational methods used in this work fail in the case of nonaromatic Y66S and Y66L protein chromophores, which may be related to a significant contribution of double excitations to their excited-state wavefunctions. These results provide an important benchmark of the reliability of the computational methods as applied to GFP chromophore analogues and lays a foundation for the computational design of GFP variants with improved properties for use in biological imaging.
由于维多利亚多管水母绿色荧光蛋白（GFP）能够在其三级结构范围内自行形成可见波长的发色团，它在生物研究中推动了重组融合蛋白荧光成像的日益普及。激发或发射波长最大值改变的GFP工程变体有助于扩大GFP的应用范围。GFP变体的工程改造通常是通过对发色团结构和/或其环境进行基因修饰来凭经验完成的，以便找到具有新光物理性质的变体。如果通过发色团基态和激发态性质及动力学的计算研究来增强或指导，那么识别改良变体的过程可能会得到极大的促进。为了实现这一目标，我们现在报告对一系列经过实验验证的工程GFP发色团类似物的吸光度最大值预测计算方法的全面研究。实验数据集由10种化学性质不同的GFP发色团类似物的吸收最大值组成，包括一个先前未报道的Y66D变体，这些数据是在相同的变性条件下测量的。对于每个发色团类似物，使用单激发组态相互作用（CIS）、含双取代微扰校正的CIS [CIS(D)]以及含溶剂效应的含时密度泛函理论（TD DFT）（使用几种密度泛函并采用极化连续介质模型）计算激发能和振子强度。实验结果与计算结果的比较表明，尝试的所有方法在定量一致性方面普遍较差。然而，计算得到的激发能与实验激发能之间可以得到良好的线性相关性（R2> 0.9）。使用纯密度泛函通过TD DFT获得的振子强度也与实验值有很好的相关性。有趣的是，这项工作中使用的大多数计算方法在非芳香族Y66S和Y66L蛋白发色团的情况下都失败了，这可能与双激发对其激发态波函数的显著贡献有关。这些结果为应用于GFP发色团类似物的计算方法的可靠性提供了重要基准，并为设计用于生物成像的具有改进性质的GFP变体奠定了基础。
