Quantitation of protein-protein interactions by thermal stability shift analysis
Quantitation of protein-protein interactions by thermal stability shift analysis
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DOI：
10.1002/pro.674
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发表时间：
2011-08-01
期刊：
影响因子：
8
通讯作者：
Hellinga, Homme W.
中科院分区：
文献类型：
--
作者：
Layton, Curtis J.;Hellinga, Homme W.
热稳定性变化分析是检查蛋白质结合相互作用的有力方法。我们证明，在某些情况下，蛋白质-蛋白质相互作用可以通过监测热稳定性的变化，使用热力学模型和数据分析方法在这项工作中提出的量化。该方法依赖于使用报告蛋白质变性的荧光染料如SYPRO橙子从热解折叠实验中测定蛋白质稳定性。数据收集是快速和直接使用现成的实时聚合酶链反应仪器。我们提出了一种方法，用于分析对应于每个合作伙伴的展开转换，以提取蛋白质之间的相互作用的亲和力。该方法不需要构建包含解离常数的滴定系列。在热位移实验中，根据去折叠转变中点的亲和力和浓度依赖性位移，获得了不同温度下的蛋白质稳定性数据。因此，亲和力的温度依赖性的治疗是固有的这种方法，并在这项研究中开发。我们使用麦芽糖结合蛋白（MBP）和热稳定的合成锚蛋白重复蛋白（Off 7）之间的相互作用作为实验测试案例，因为它们的解折叠转换重叠最小。我们发现MBP被Off 7显著稳定。高实验通量通过样品平行化以及在单一配偶体浓度下提取定量结合信息的能力来实现。在一个单一的实验中，我们能够量化的亲和力的一系列丙氨酸突变体，涵盖了广泛的亲和力（类似于100纳米到类似于100 μ M）。
Thermal stability shift analysis is a powerful method for examining binding interactions in proteins. We demonstrate that under certain circumstances, protein-protein interactions can be quantitated by monitoring shifts in thermal stability using thermodynamic models and data analysis methods presented in this work. This method relies on the determination of protein stabilities from thermal unfolding experiments using fluorescent dyes such as SYPRO Orange that report on protein denaturation. Data collection is rapid and straightforward using readily available real-time polymerase chain reaction instrumentation. We present an approach for the analysis of the unfolding transitions corresponding to each partner to extract the affinity of the interaction between the proteins. This method does not require the construction of a titration series that brackets the dissociation constant. In thermal shift experiments, protein stability data are obtained at different temperatures according to the affinity-and concentration-dependent shifts in unfolding transition midpoints. Treatment of the temperature dependence of affinity is, therefore, intrinsic to this method and is developed in this study. We used the interaction between maltose-binding protein (MBP) and a thermostable synthetic ankyrin repeat protein (Off7) as an experimental test case because their unfolding transitions overlap minimally. We found that MBP is significantly stabilized by Off7. High experimental throughput is enabled by sample parallelization, and the ability to extract quantitative binding information at a single partner concentration. In a single experiment, we were able to quantify the affinities of a series of alanine mutants, covering a wide range of affinities (similar to 100 nM to similar to 100 mu M).