Hennig Andreas, Nau Werner M
Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Bremen, Germany.
Institute of Chemistry of New Materials, School of Biology/Chemistry, Universität Osnabrück, Osnabrück, Germany.
Front Chem. 2020 Sep 10;8:806. doi: 10.3389/fchem.2020.00806. eCollection 2020.
We report the use of the macrocyclic host cucurbit[7]uril (CB7) as a supramolecular additive in nanosecond time-resolved fluorescence (Nano-TRF) assays for proteases to enhance the discrimination of substrates and products and, thereby, the sensitivity. A peptide substrate was labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a long-lived (>300 ns) fluorescent probe and 3-nitrotyrosine was established as a non-fluorescent fluorescence resonance energy transfer (FRET) acceptor that acts as a "dark quencher." The substrate was cleaved by the model proteases trypsin and chymotrypsin and the effects of the addition of CB7 to the enzyme assay mixture were investigated in detail using UV/VIS absorption as well as steady-state and time-resolved fluorescence spectroscopy. This also allowed us to identify the DBO and nitrotyrosine residues as preferential binding sites for CB7 and suggested a hairpin conformation of the peptide, in which the guanidinium side chain of an arginine residue is additionally bound to a vacant ureido rim of one of the CB7 hosts.
我们报道了将大环主体葫芦[7]脲（CB7）用作蛋白酶纳秒时间分辨荧光（Nano-TRF）测定中的超分子添加剂，以增强对底物和产物的区分能力，从而提高灵敏度。一种肽底物用2,3-二氮杂双环[2.2.2]辛-2-烯（DBO）标记作为长寿命（>300 ns）荧光探针，3-硝基酪氨酸被确定为作为“暗猝灭剂”的非荧光荧光共振能量转移（FRET）受体。该底物被模型蛋白酶胰蛋白酶和胰凝乳蛋白酶切割，并使用紫外/可见吸收以及稳态和时间分辨荧光光谱详细研究了向酶测定混合物中添加CB7的效果。这也使我们能够确定DBO和硝基酪氨酸残基是CB7的优先结合位点，并表明该肽呈发夹构象，其中精氨酸残基的胍基侧链额外结合到其中一个CB7主体的空脲基边缘。
