Using a novel dual fluorescence quenching assay for measurement of tryptophan depth within lipid Bilayers to determine hydrophobic α-helix locations within membranes
Using a novel dual fluorescence quenching assay for measurement of tryptophan depth within lipid Bilayers to determine hydrophobic α-helix locations within membranes
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DOI：
10.1021/bi026696l
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发表时间：
2003-03-25
期刊：
影响因子：
2.9
通讯作者：
London, E
中科院分区：
文献类型：
--
作者：
Caputo, GA;London, E
介绍了一种测定膜插入多肽中色氨酸残基深度的新荧光法。用丙烯酰胺和10-十二烷（10-DN）猝灭Trp来测量Trp深度。跨膜螺旋与色氨酸残基在不同的位置（因此位于不同的深度在脂质双层）被用来校准的方法。结果发现，丙烯酰胺淬灭色氨酸接近双层表面更强烈地比它淬灭深埋色氨酸，而10-DN淬灭色氨酸接近双层中心更强烈地比色氨酸接近表面。发现丙烯酰胺淬灭与10-DN淬灭的比率与膜中Trp的深度几乎呈线性关系。还发现，使用这些猝灭剂可以检测共存的浅插入和深插入的含Trp多肽群体。在这样的混合人群的存在下，丙烯酰胺诱导大的荧光发射λ（最大值）的蓝移，而10-DN诱导大的红移。在一个更均匀的人口淬灭诱导的变化被认为是最小的。双猝灭剂分析可用于区分疏水螺旋与跨膜取向的那些位于接近双层表面，当应用于一些不同的肽，揭示了疏水螺旋行为的新方面。
A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polypeptides is introduced. Quenching of Trp by acrylamide and 10-doxylnonadecane (10-DN) was used to measure Trp depth. Transmembrane helices with Trp residues at varying positions (and thus locating at different depths in lipid bilayers) were used to calibrate the method. It was found that acrylamide quenches Trp close to the bilayer surface more strongly than it quenches deeply buried Trp, while 10-DN quenches Trp close to the center of the bilayer more strongly than Trp close to the surface. The ratio of acrylamide quenching to that of 10-DN was found to be nearly linearly dependent on the depth of Trp in a membrane. It was also found that it was possible to detect coexisting shallowly and deeply inserted populations of Trp-containing polypeptides using these quenchers. In the presence of such mixed populations, acrylamide induced large blue shifts in fluorescence emission lambda(max) whereas 10-DN induced large red shifts. In a more homogeneous population quencher-induced shifts were found to be minimal. Dual quencher analysis can be used to distinguish hydrophobic helices with a transmembrane orientation from those located close to the bilayer surface and, when applied to a number of different peptides, revealed novel aspects of hydrophobic helix behavior.