期刊
JOURNAL OF PHYSICAL CHEMISTRY B
卷 124, 期 47, 页码 10653-10662出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jpcb.0c06865
关键词
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资金
- University of Pennsylvania
- National Science Foundation [NSF DGE-1321851, NSF CHE-1708759]
- Parkinson's Disease Foundation [PF-RVSA-SFW-1754]
- University of Pennsylvania Center for Undergraduate and Research Fellowships
- Roy and Diana Vagelos Science Challenge Award
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 of p-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 occurs via Forster 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.
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