提高膜蛋白热稳定性的简易筛选方法。
Simple screening method for improving membrane protein thermostability.
机构信息
The Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.
出版信息
Methods. 2011 Dec;55(4):324-9. doi: 10.1016/j.ymeth.2011.07.008. Epub 2011 Aug 5.
Abstract
Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this technique we were able to assess and improve the thermostability of several membrane proteins. These conditions were in turn used to assist purification, to identify protein ligand and to improve crystal quality.
摘要
生化和生物物理分析整体膜蛋白通常需要单分散和稳定的蛋白质样品。在这里，我们描述了一种通过使用分析尺寸排阻色谱法在去污剂中测量其熔点来测定蛋白质热稳定性的方法。这种定量方法可用于筛选稳定蛋白质的化合物和条件。通过该技术，我们能够评估和提高几种膜蛋白的热稳定性。这些条件反过来又被用于辅助蛋白质的纯化、鉴定蛋白质配体和改善晶体质量。
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1
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2
Evolution of three human GPCRs for higher expression and stability.三种人类 GPCR 为了更高的表达和稳定性而进化。
J Mol Biol. 2011 May 13;408(4):599-615. doi: 10.1016/j.jmb.2011.02.051. Epub 2011 Mar 3.
3
The structural basis for agonist and partial agonist action on a β(1)-adrenergic receptor.激动剂和部分激动剂在β(1)-肾上腺素能受体上作用的结构基础。
Nature. 2011 Jan 13;469(7329):241-4. doi: 10.1038/nature09746.
4
Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures.为提高高分辨率 X 射线结构的通量而对膜蛋白去污剂稳定性进行基准测试。
Structure. 2011 Jan 12;19(1):17-25. doi: 10.1016/j.str.2010.12.001.
5
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