Institute of Theoretical and Computational Chemistry, Key Laboratory of Mesoscopic Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, People's Republic of China.
J Phys Chem A. 2011 Sep 15;115(36):10129-35. doi: 10.1021/jp2033609. Epub 2011 Aug 24.
In red fluorescent proteins such as DsRed, an acylimine is formed from the Phe65-Gln66 linkage in GFP-like immature form, while it shows a cis configuration in its mature state. To date, the relationship between acylimine formation and trans-cis isomerization is still unresolved. We have calculated bond rotation profiles for mature and immature chromophores within the protein using our own n-layered integrated molecular orbital and molecular mechanism (ONIOM) approach. The results suggested that the isomerization is barrierless in acylimine formed in the mature state, suggesting that the acylimine formation precedes the trans-cis isomerization in DsRed chromophores. Further decomposition analysis of electrostatic contributions from individual residues has identified several residues and a specific water molecule which could play key roles in controlling the rate of the trans-cis isomerization of peptide bond in immature GFP-like protein. The results also highlight the importance of Gln66-like of tripeptide motif (chromophore) in the maturation of red fluorescent proteins. In view of the considerable interest in developing red fluorescent proteins for numerous biotechnological applications, these results should be useful for design of novel fluorescent proteins.
在红色荧光蛋白（如 DsRed）中，酰亚胺是由 GFP 样未成熟形式中的 Phe65-Gln66 键形成的，而在其成熟状态下则呈现顺式构型。迄今为止，酰亚胺形成与顺反异构之间的关系仍未得到解决。我们使用自己的 n 层综合分子轨道和分子机制（ONIOM）方法计算了蛋白质中成熟和未成熟发色团的键旋转轮廓。结果表明，在成熟状态下形成的酰亚胺中，异构化是无阻碍的，这表明 DsRed 发色团中的酰亚胺形成先于顺反异构化。对来自单个残基的静电贡献的进一步分解分析确定了几个残基和一个特定的水分子，它们可能在控制未成熟 GFP 样蛋白中肽键顺反异构化的速率方面发挥关键作用。这些结果还强调了三肽基序（发色团）中 Gln66 样残基在红色荧光蛋白成熟过程中的重要性。鉴于人们对开发红色荧光蛋白用于众多生物技术应用的浓厚兴趣，这些结果对于设计新型荧光蛋白应该是有用的。
