利用法蘭克-康登擬合綠色螢光蛋白質發色團的吸收與螢光光譜

Abstract

綠色螢光蛋白質的發色團是經過分子內的一些反應而形成，由第65號的絲胺酸(serine-65)、第66號的酪胺酸(tyrosin-66)和第67號的甘胺酸(glycine-67)組成主要的結構，而紅色螢光蛋白質的發色團則是由第66號的谷氨醯胺(glutamine-66)、第67號的酪胺酸(tyrosin-67)和第68號的甘胺酸(glycine-68)所組成。綠色以及紅色螢光蛋白質的發色團同時具有4-hydroxybenzylideneimidazolinone的部分，但imidazolinone ring所連接的官能基則不同。將兩者發色團的吸收性質互相比較，若所連接的官能基是Π共軛系統，將會造成明顯的紅位移。 在本研究工作裡，我們主要使用法蘭克-康登擬合(Franck-Condon simulation)綠色和紅色螢光蛋白質之發色團的吸收以及螢光光譜，而法蘭克-康登擬合的過程包含了均勻與非均勻加寬和簡諧與非簡諧的討論。在基態的計算方面，則使用了三種不同交換−相關效應的密度泛函(B3LYP, B3LYP-35, and BHandHLYP)和Hartree-Fock方法，結合使用極化連續模型來計算溶劑效應。而在激發態的計算方面，則必須基於使用相同層級的ab initio方法，例如含時密度泛函理論(TD-DFT)對應密度泛函理論(DFT)、單電子激發組態交互作用(CIS)則對應Hartree-Fock方法。 本研究成果成功的論述了HBDI的吸收和螢光光譜，不論是在譜峰的位置，抑或是光譜的寬度，與實驗光譜相比都具有很好的一致性。

The GFP chromophore is formed by the intramolecular reaction from tripeptide serine-65 (Ser65), tyrosin-66 (Tyr66), and glycine-67 (Gly67) in the native protein for the primary structure while the chromophore in DsRed is also formed from the three amino acids which are glutamine-66 (Gln66), tyrosin-67 (Tyr67), and glycine-68 (Gly68) for the primary structure. Both the GFP and DsRed chromophores share the same 4-hydroxybenzylideneimidazolinone core, but the linkages at the imidazolinone ring are different. The extension of the conjugated Π-system in the chromophore may cause the significant red shift of the DsRed absorption properties compared to those of GFP. In the present work, we demonstrate the absorption and fluorescence spectra of the GFP and DsRed chromophore in Franck-Condon simulation with including homogenous broadening, inhomogenous broadening, harmonic, and anharmonic discussions. Three different exchange-correlation functionals (B3LYP, B3LYP-35, and BHandHLYP) of density functional theory (DFT) and Hartree-Fock (HF) method with and without the polarizable continuum model (PCM) were used for the ground state. At the same level of ab initio methods for the excited states, time-dependent density functional theory (TD-DFT) verse DFT and configuration interaction singles (CIS) verse HF, respectively. The results presented in this study demonstrate that peak position and spectral widths of both absorption and fluorescence spectra for HBDI agree with experimental observations.