کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1944187 | 1053188 | 2014 | 7 صفحه PDF | دانلود رایگان |
• Membrane penetration is characterized by depth-dependent fluorescence quenching.
• Distribution analysis method is used to extract quantitative information.
• Manuscript reviews principles and applications of distribution analysis.
• Molecular dynamics simulations are used to validate method's assumptions.
• Intensity and lifetime measurements are used to refine membrane penetration depth.
Depth-dependent fluorescence quenching by lipid-attached quenchers (e.g., bromine atoms and doxyl groups) is an important tool for determining the penetration of proteins and peptides into lipid bilayers. Extracting quantitative information and accurate calculations of the depth of the fluorophore are complicated by thermal disorder, resulting in broad distributions of the transverse positions of both quenchers and fluorophores. Twenty-one years ago a methodology called distribution analysis (DA) was introduced, based on the emerging view of the complexity of the transverse organization of lipid bilayer structure. The method is aimed at extracting quantitative information on membrane penetration, such as position and width of fluorophore's distribution along the depth coordinate and its exposure to the lipid phase. Here we review recent progress in refining the DA method and illustrate its applications to protein–membrane interactions. We demonstrate how basic assumptions of the DA approach can be validated using molecular dynamics simulations and how the precision of depth determination is improved by applying a new protocol based on a combination of steady-state and time-resolved fluorescence quenching. Using the example of the MPER fragment of the membrane-spanning domain of the HIV-1 gp41 fusion protein, we illustrate how DA applications and computer simulations can be used together to reveal the molecular organization of a protein–membrane complex. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.
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Journal: Biochimica et Biophysica Acta (BBA) - Biomembranes - Volume 1838, Issue 9, September 2014, Pages 2289–2295