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通过三检测尺寸排阻色谱法表征蛋白质/去污剂复合物

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography.

作者信息

Gimpl Katharina, Klement Jessica, Keller Sandro

机构信息

Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.

出版信息

Biol Proced Online. 2016 Feb 15;18:4. doi: 10.1186/s12575-015-0031-9. eCollection 2016.

DOI:10.1186/s12575-015-0031-9
PMID:26880869
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4753644/
Abstract

BACKGROUND

In vitro investigations of membrane proteins usually depend on detergents for protein solubilisation and stabilisation. The amount of detergent bound to a membrane protein is relevant to successful experiment design and data analysis but is often unknown. Triple-detection size-exclusion chromatography enables simultaneous separation of protein/detergent complexes and protein-free detergent micelles and determination of their molar masses in a straightforward and absolute manner. Size-exclusion chromatography is used to separate different species, while ultraviolet absorbance, static light scattering, and refractive index measurements allow molar mass determination of protein and detergent components.

RESULTS

We refined standard experimental and data-analysis procedures for challenging membrane-protein samples that elude routine approaches. The general procedures including preparatory steps, measurements, and data analysis for the characterisation of both routine and complex samples in difficult solvents such as concentrated denaturant solutions are demonstrated. The applicability of the protocol but also its limitations and possible solutions are discussed, and an extensive troubleshooting section is provided.

CONCLUSIONS

We established and validated a protocol for triple-detection size-exclusion chromatography that enables the inexperienced user to perform and analyse measurements of well-behaved protein/detergent complexes. More experienced users are provided with an example of a more sophisticated analysis procedure allowing mass determination under challenging separation conditions.

摘要

背景

膜蛋白的体外研究通常依赖于去污剂来溶解和稳定蛋白质。与膜蛋白结合的去污剂的量与成功的实验设计和数据分析相关，但往往是未知的。三检测尺寸排阻色谱法能够同时分离蛋白质/去污剂复合物和无蛋白质的去污剂胶束，并以直接和绝对的方式测定它们的摩尔质量。尺寸排阻色谱法用于分离不同的物种，而紫外吸收、静态光散射和折射率测量则允许测定蛋白质和去污剂成分的摩尔质量。

结果

我们改进了针对难以用常规方法处理的具有挑战性的膜蛋白样品的标准实验和数据分析程序。展示了用于表征常规样品和复杂样品的一般程序，包括在诸如浓变性剂溶液等难溶性溶剂中的制备步骤、测量和数据分析。讨论了该方案的适用性及其局限性和可能的解决方案，并提供了一个广泛的故障排除部分。

结论

我们建立并验证了一种三检测尺寸排阻色谱法的方案，使缺乏经验的用户能够进行并分析行为良好的蛋白质/去污剂复合物的测量。为经验更丰富的用户提供了一个更复杂的分析程序示例，该程序允许在具有挑战性的分离条件下进行质量测定。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/59e7697e6b6c/12575_2015_31_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/7d176ebc6b69/12575_2015_31_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/a080314565e5/12575_2015_31_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/cbd218c7f43c/12575_2015_31_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/7776a7f2fb52/12575_2015_31_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/3bf24f53c303/12575_2015_31_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/59e7697e6b6c/12575_2015_31_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/7d176ebc6b69/12575_2015_31_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/a080314565e5/12575_2015_31_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/cbd218c7f43c/12575_2015_31_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/7776a7f2fb52/12575_2015_31_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/3bf24f53c303/12575_2015_31_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a4b/4753644/59e7697e6b6c/12575_2015_31_Fig6_HTML.jpg

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