Calibration of Distribution Analysis of the Depth of Membrane Penetration Using Simulations and Depth-Dependent Fluorescence Quenching.
Calibration of Distribution Analysis of the Depth of Membrane Penetration Using Simulations and Depth-Dependent Fluorescence Quenching.
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DOI：
10.1007/s00232-014-9709-1
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发表时间：
2015-06
影响因子：
2.4
通讯作者：
Ladokhin, Alexey S.
中科院分区：
文献类型：
--
作者：
Kyrychenko, Alexander;Rodnin, Mykola V.;Ladokhin, Alexey S.
关键词：
膜渗透深度的测定为研究膜蛋白折叠和蛋白脂相互作用提供了重要信息。在这里，我们使用分子动力学（MD）模拟和深度依赖的荧光猝灭相结合来校准提取膜穿透定量信息的方法。为了研究荧光标记在脂质双分子层中的浸没深度，我们研究了7-硝基苯-2-氧杂-1,3-二唑（NBD）在加入到POPC双分子层的NBD- pe中附着在脂质头基上的情况。通过测量稳态和时间分辨荧光猝灭来估计NBD的浸泡深度，其中自旋标记的脂质共掺入脂质囊泡中。使用了六种不同的自旋标记脂质：一种是头基团连接的Tempo探针（Tempo- pc），五种是酰基链标记的n-羟基部分（n-羟基- pc，其中n为链标记位置，分别为5,7,10,12和14）。Stern-Volmer分析表明，NBD在膜中的猝灭可通过静态和动态碰撞猝灭过程发生。利用分布分析的方法，估计NBD部分横向分布的浸没深度和表观半宽度分别为14.9 Å和6.7 Å。在POPC双层中NBD-PE脂质的原子MD模拟独立验证了这一位置（14.4 Å）。此外，我们还证明了染料和猝灭剂分布之间的横向重叠积分的MD模拟可以用于深度相关猝灭曲线的适当分析。最后，我们通过确定白喉毒素t结构域位点选择性标记突变体的膜渗透来说明这种方法的应用。
Determination of the depth of membrane penetration provides important information for studies of membrane protein folding and protein-lipid interactions. Here we use a combination of molecular dynamics (MD) simulations and depth-dependent fluorescence quenching to calibrate the methodology for extracting quantitative information on membrane penetration. In order to investigate the immersion depth of the fluorescent label in lipid bilayer, we studied 7-nitrobenz-2-oxa-1,3-diazole (NBD) attached to the lipid headgroup in NBD-PE incorporated into POPC bilayer. The immersion depth of NBD was estimated by measuring steady-state and time-resolved fluorescence quenching with spin-labeled lipids co-incorporated into lipid vesicles. Six different spin-labeled lipids were utilized: one with headgroup-attached Tempo probe (Tempo-PC) and five with acyl-chain-labeled n-Doxyl moieties (n-Doxyl-PC where n is a chain labeling position equal to 5, 7, 10, 12, and 14, respectively). The Stern-Volmer analysis revealed that NBD quenching in membranes occurs by both static and dynamic collisional quenching processes. Using the methodology of Distribution Analysis, the immersion depth and the apparent half-width of the transversal distributions of the NBD moiety were estimated to be 14.9 Å and 6.7 Å from the bilayer center. This position is independently validated by atomistic MD simulations of NBD-PE lipids in a POPC bilayer (14.4 Å). In addition, we demonstrate that MD simulations of the transverse overlap integrals between dye and quencher distributions can be used for proper analysis of the depth-dependent quenching profile. Finally, we illustrate the application of this methodology by determining membrane penetration of site-selectively labeled mutants of diphtheria toxin T-domain.
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DOI：
10.1016/0005-2736(88)90118-6
发表时间：
1988-02-08
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
CHATTOPADHYAY, A;LONDON, E
通讯作者：
LONDON, E
影响因子：
3.3
作者：
Kyrychenko, Alexander;Wu, Feiyue;Thummel, Randolph P.;Waluk, Jacek;Ladokhin, Alexey S.
通讯作者：
Ladokhin, Alexey S.
影响因子：
3.9
作者：
Armstrong, VT;Brzustowicz, MR;Stillwell, W
通讯作者：
Stillwell, W
影响因子：
2.9
作者：
CHATTOPADHYAY, A;LONDON, E
通讯作者：
LONDON, E
影响因子：
3.3
作者：
Filipe, Hugo A. L.;Moreno, Maria Joao;Loura, Luis M. S.
通讯作者：
Loura, Luis M. S.