Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.
Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.
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使用荧光染料和热展开对膜和可溶性蛋白质中的自由半胱氨酸进行定量。
DOI：
10.1038/nprot.2013.128
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发表时间：
2013-11
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
半胱氨酸是选择性标记蛋白质的一个非常有用的位置，在许多应用中，包括用电子顺磁共振(EPR)、荧光光谱和医学成像研究溶液中的蛋白质结构。这些应用对定量数据的需求意味着确定半胱氨酸标记的范围是重要的。标记的效率对蛋白质中半胱氨酸的3D环境很敏感。在不能通过光学或磁光谱学直接测量标记或修饰的情况下，例如，在半胱氨酸修饰为脱氢丙氨酸的情况下，很难评估标记效率。我们描述了一种确定半胱氨酸残基修饰效率的简单方法，该方法基于以前用于确定膜蛋白稳定性的方法。该分析涉及热展开的蛋白质与硫醇特异的香豆素荧光团之间的反应，该荧光团只有在与硫醇结合时才是荧光的。在染料存在的情况下，监测蛋白质热变性过程中的荧光，确定出现最大荧光的温度；这个温度因蛋白质而异。比较修饰的、未修饰的(阳性对照)和不含半胱氨酸的蛋白质(阴性对照)在确定的温度下的荧光强度，可以对游离半胱氨酸进行定量。我们对定点自旋标记和脱氢丙氨酸的形成进行了量化。该方法依赖于常见的荧光96孔板读取器，该读取器在1.5h内快速筛选大量样品，并使用<；100μg材料。该方法对可溶性和洗涤剂增溶的膜蛋白都是稳健的。
Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.
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影响因子：
16.8
作者：
通讯作者：
--
影响因子：
15
作者：
Endeward, Burkhard;Butterwick, Joel A.;MacKinnon, Roderick;Prisner, Thomas F.
通讯作者：
Prisner, Thomas F.
影响因子：
3.4
作者：
Liu, Wei;Hanson, Michael A.;Cherezov, Vadim
通讯作者：
Cherezov, Vadim
影响因子：
4.8
作者：
Rasmussen, Tim;Edwards, Michelle D.;Booth, Ian R.
通讯作者：
Booth, Ian R.
DOI：
10.1074/jbc.m109.065458
发表时间：
2010-01-22
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
White GF;Schermann SM;Bradley J;Roberts A;Greene NP;Berks BC;Thomson AJ
通讯作者：
Thomson AJ