Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA.
J Am Chem Soc. 2012 Apr 11;134(14):6088-91. doi: 10.1021/ja3005094. Epub 2012 Apr 3.
Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond-a single atom substitution of the peptide backbone-can quench fluorophores that are red-shifted from intrinsic protein fluorescence, such as acridone. Using steady-state and fluorescence lifetime measurements, we further demonstrate that this quenching occurs through a dynamic electron-transfer mechanism. In a proof-of-principle experiment, we apply this technique to monitor unfolding in a model peptide system, the villin headpiece HP35 fragment. Thioamide analogues of the natural amino acids can be placed in a variety of locations in a protein sequence, allowing one to make a large number of measurements to model protein folding.
在色氨酸和酪氨酸存在的情况下能够被选择性激发的荧光探针对在研究蛋白质构象变化方面非常有用。然而，这些探针对的大小可能会限制它们在蛋白质序列的小部分中被结合，因为在这些部位探针对的结合对二级和三级结构的影响是可以被容忍的。我们的研究结果表明，硫酰胺键——肽主链上单个原子的取代——可以猝灭从蛋白质固有荧光红移的荧光团，如吖啶酮。通过稳态和荧光寿命测量，我们进一步证明这种猝灭是通过动态电子转移机制发生的。在一个原理验证实验中，我们将该技术应用于监测模型肽系统——肌球蛋白头部 HP35 片段的展开。天然氨基酸的硫酰胺类似物可以被放置在蛋白质序列的各种位置，这允许人们进行大量的测量以模拟蛋白质折叠。
