利用定位自旋标记(SDSL)测定可溶性蛋白的脉冲EPR距离

Q1 Biochemistry, Genetics and Molecular Biology

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI:10.1002/0471140864.ps1717s74

Ian Mitchelle S. de Vera, Mandy E. Blackburn, Luis Galiano, Gail E. Fanucci

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引用次数: 19

摘要

脉冲电子顺磁共振(EPR)在结构生物学领域的复兴主要是由于双电子-电子共振(DEER)技术在距离测量方面的最新进展。本单元以HIV-1蛋白酶为模型，通过位点定向自旋标记(SDSL)对可溶性蛋白中的工程半胱氨酸残基进行基于epr的距离测量。为了阐明蛋白质的构象变化，对实验方案进行了优化，并利用现有的数据分析程序推导了距离分布曲线。本文还概述了伪影抑制的实验考虑、样品制备和误差分析。咕咕叫。Protoc。蛋白质科学，74:17.17.1-17.17.29。©2013 by John Wiley &儿子,Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

Pulsed EPR Distance Measurements in Soluble Proteins by Site-Directed Spin Labeling (SDSL)

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Pulsed EPR Distance Measurements in Soluble Proteins by Site-Directed Spin Labeling (SDSL)

The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron-electron resonance (DEER) technique. This unit focuses on EPR-based distance measurements by site-directed spin labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV-1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance-distribution profiles. Experimental considerations, sample preparation, and error analysis for artifact suppression are also outlined herein. Curr. Protoc. Protein Sci. 74:17.17.1-17.17.29. © 2013 by John Wiley & Sons, Inc.

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Current Protocols in Protein Science Biochemistry, Genetics and Molecular Biology-Biochemistry

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期刊介绍： With the mapping of the human genome, more and more researchers are exploring protein structures and functions in living organisms. Current Protocols in Protein Science provides protein scientists, biochemists, molecular biologists, geneticists, and others with the first comprehensive suite of protocols for this growing field.