How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?
How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?
复制标题
DOI：
10.1006/abio.2000.4773
复制
发表时间：
2000-10-15
影响因子：
2.9
通讯作者：
White, SH
中科院分区：
文献类型：
--
作者：
Ladokhin, AS;Jayasinghe, S;White, SH
色氨酸荧光是研究蛋白质结构和功能的有力工具，尤其是膜活性蛋白质和多肽，是研究蛋白质和多肽与囊泡单层模型膜相互作用的最常用工具。然而，与泡状膜系统相关的高光散射带来了特殊的挑战。与大的单层囊泡(LUV)相比，超声产生的小的单层囊泡(Sw)具有较小的光散射，因此被广泛应用于膜模型。不幸的是，与LW不同的是，Sw是亚稳态的，因此不适合用于平衡热力学测量。我们介绍了在LUV或SW中准确测定色氨酸(Trp)荧光的简单和容易实现的实验方法，具体地说，我们证明了在高达6 mm的LUV存在下，Trp光谱可以获得与单独在缓冲液中获得的光谱几乎相同的光谱，这避免了使用SW。我们展示了如何使用这种光谱的宽度和峰位来评价多肽的膜构象和穿透的异质性，最后，我们展示了如何使用参考荧光团来校正强度测量，以便准确地测定多肽分配到膜中的能量。(C)2000年学术出版社。
Tryptophan fluorescence is a powerful tool for studying protein structure and function, especially membrane-active proteins and peptides, It is arguably the most frequently used tool for examining the interactions of proteins and peptides with vesicular unilamellar model membranes. However, high light scattering associated with vesicular membrane systems presents special challenges. Because of their reduced light scattering compared to large unilamellar vesicles (LUV), small unilamellar vesicles (SW) produced by sonication are widely used membrane models. Unfortunately, SW, unlike LW, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. We present simple and easily implemented experimental procedures for the accurate determination of tryptophan (Trp) fluorescence in either LUV or SW, Specifically, we show that Trp spectra can be obtained in the presence of up to 6 mM LUV that are virtually identical to spectra obtained in buffer alone, which obviates the use of SW, We show how the widths and peak positions of such spectra can be used to evaluate the heterogeneity of the membrane conformation and penetration of peptides, Finally, we show how to use a reference fluorophore for the correction of intensity measurements so that the energetics of peptide partitioning into membranes can be accurately determined. (C) 2000 Academic Press.