Bimolecular quenching of tryptophan fluorescence in a membrane protein: Evolution of local solvation and environment during folding into a bilayer
Bimolecular quenching of tryptophan fluorescence in a membrane protein: Evolution of local solvation and environment during folding into a bilayer
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DOI：
10.1016/j.saa.2021.119919
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发表时间：
2021-05-15
影响因子：
4.4
通讯作者：
Kim, Judy E.
中科院分区：
文献类型：
--
作者：
Asamoto, DeeAnn K.;Kozachenko, Ivan A.;Kim, Judy E.
荧光光谱（包括 Stern-Volmer 猝灭）是研究蛋白质动力学的重要工具。使用丙烯酰胺猝灭剂进行双分子荧光猝灭，探究膜蛋白、外膜蛋白 A (OmpA) 折叠反应成脂质双层期间蛋白质溶剂化的变化。六个单色氨酸 OmpA 突变体（W7、W15、W57、W102、W129 和 W143）允许在跨膜 β-桶结构域内的不同位置进行位点特异性研究。结合了静态和动态成分的作用域猝灭模型产生了 Stern-Volmer 猝灭常数 K-D，OmpA 在 8.0 M 尿素中变性，在 0.5 M 尿素中聚集，吸附到小单层囊泡 (SUV) 上，并在 SUV 中折叠 (t = 6 小时)。平均K-D值是K-D（变性）（6.4 M-1）> K-D（聚集）（5.9 M-1）> K-D（吸附）（1.9 M-1）> K-D（折叠）（0.6 M-1）。了解了不存在猝灭剂时的荧光寿命，得出了双分子猝灭常数 kq；在 SUV 折叠反应期间（t = 0 小时至 t = 6 小时），k(q)（以及 K-D）的演变揭示了去溶剂化时间尺度，tau(desolv) 为 41-46 分钟（W7、W15、W57、W102）、27 分钟（W129）和 15 分钟（W143）。折叠过程中 lambda(max) 的演化揭示了所有突变体的快成分和慢成分，tau(快)（环境）和 tau(慢)（环境）分别为 7-13 分钟和 25-84 分钟。对于五个面向脂质的突变体（W7、W15、W57、W129 和 W143），总体趋势是快速环境7 13 分钟THORN < tau(desolv)（15 - 46 分钟）
Fluorescence spectroscopy, including Stern-Volmer quenching, is a valuable tool for the study of protein dynamics. Changes in protein solvation during the folding reaction of a membrane protein, Outer membrane protein A (OmpA), into lipid bilayers was probed with bimolecular fluorescence quenching with acrylamide quencher. Six single-tryptophan OmpA mutants (W7, W15, W57, W102, W129, and W143) allowed for site-specific investigations at varying locations within the transmembrane beta-barrel domain. A sphere-of-action quenching model that combines both static and dynamic components gave rise to Stern-Volmer quenching constants, K-D, for OmpA denatured in 8.0 M urea, aggregated in 0.5 M urea, adsorbed onto small unilamellar vesicles (SUVs), and folded in SUVs (t = 6 hrs). The average K-D values were K-D(denatured) (6.4 M-1) > K-D(aggregated) (5.9 M-1) > K-D(adsorbed) (1.9 M-1) > K-D(folded) (0.6 M-1). With knowledge of the fluorescence lifetimes in the absence of quencher, the bimolecular quenching constants, kq, were derived; the evolution of k(q) (and therefore K-D)during the folding reaction into SUVs (t = 0 hr to t = 6 hrs) revealed desolvation timescales, tau(desolv) of 41-46 min (W7, W15, W57, W102), 27 min (W129), and 15 min (W143). The evolution of lambda(max) during folding revealed fast and slow components, tau(fast)(environment) and tau(slow)(environment) of 7-13 min and 25-84 min, respectively, for all mutants. For the five lipid-facing mutants (W7, W15, W57, W129, and W143), the general trend was sfast environmento7 13 minTHORN < tau(desolv) (15 - 46 min