Improved Modeling of Thioamide FRET Quenching by Including Conformational Restriction and Coulomb Coupling
Improved Modeling of Thioamide FRET Quenching by Including Conformational Restriction and Coulomb Coupling
复制标题
通过构象限制和库仑耦合改进硫代酰胺 FRET 淬灭模型
DOI：
10.1021/acs.jpcb.0c06865
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Petersson, E. James
中科院分区：
文献类型：
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作者：
Yoon, Jimin;Ferrie, John J.;Petersson, E. James
已显示含硫代酰胺的氨基酸通过不同的机制淬灭广泛的荧光团。在这里，我们定量分析硫代酰胺官能团淬灭对氰基苯丙氨酸（Cnf），酪氨酸（Tyr）和色氨酸（Trp）的荧光的机制。通过比较PyRosetta模拟发表的实验进行的聚脯氨酸统治者肽，我们证实了以前的研究结果，即Cnf和Tyr淬灭发生viaFo rster共振能量转移（FRET），而色氨酸淬灭发生通过一种替代机制，如德克斯特转移。此外，肽采样方案的优化以及与肽骨架和侧链连接的硫代酰胺的比较表明，与硫代酰胺部分相关的显着构象限制导致表观FRET效率对潜在构象差异的高度敏感性。此外，通过计算FRET效率的结构模型，使用各种方法，我们发现，定量准确的库仑耦合的作用是需要解释的贡献，从个别结构上的详细程度上观察到的淬灭效率。最后，我们证明，这些额外的考虑提高了我们的能力，预测硫代酰胺淬灭效率的硫代酰胺标记的肽荧光标记的钙调蛋白的变体结合过程中观察到。
Thioamide-containing amino acids have been shown to quench a wide range of fluorophores through distinct mechanisms. Here, we quantitatively analyze the mechanism through which the thioamide functional group quenches the fluorescence ofp-cyanophenylalanine (Cnf), tyrosine (Tyr), and tryptophan (Trp). By comparing PyRosetta simulations to published experiments performed on polyproline ruler peptides, we corroborate previous findings that both Cnf and Tyr quenching occursviaFörster resonance energy transfer (FRET), while Trp quenching occurs through an alternate mechanism such as Dexter transfer. Additionally, optimization of the peptide sampling scheme and comparison of thioamides attached to the peptide backbone and side chain revealed that the significant conformational restriction associated with the thioamide moiety results in a high sensitivity of the apparent FRET efficiency to underlying conformational differences. Moreover, by computing FRET efficiencies from structural models using a variety of approaches, we find that quantitative accuracy in the role of Coulomb coupling is required to explain contributions to the observed quenching efficiency from individual structures on a detailed level. Last, we demonstrate that these additional considerations improve our ability to predict thioamide quenching efficiencies observed during binding of thioamide-labeled peptides to fluorophore-labeled variants of calmodulin.