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Nanomolar Protein–Protein Interaction Monitoring with a Label-Free Protein-Probe Technique
Analytical Chemistry ( IF 7.4 ) Pub Date : 2020-11-25 , DOI: 10.1021/acs.analchem.0c02823
Salla Valtonen 1 , Emmiliisa Vuorinen 1 , Taru Kariniemi 1 , Ville Eskonen 1 , John Le Quesne 2 , Martin Bushell 3, 4 , Harri Härmä 1 , Kari Kopra 1
Affiliation  

Protein–protein interactions (PPIs) are an essential part of correct cellular functionality, making them increasingly interesting drug targets. While Förster resonance energy transfer-based methods have traditionally been widely used for PPI studies, label-free techniques have recently drawn significant attention. These methods are ideal for studying PPIs, most importantly as there is no need for labeling of either interaction partner, reducing potential interferences and overall costs. Already, several different label-free methods are available, such as differential scanning calorimetry and surface plasmon resonance, but these biophysical methods suffer from low to medium throughput, which reduces suitability for high-throughput screening (HTS) of PPI inhibitors. Differential scanning fluorimetry, utilizing external fluorescent probes, is an HTS compatible technique, but high protein concentration is needed for experiments. To improve the current concepts, we have developed a method based on time-resolved luminescence, enabling PPI monitoring even at low nanomolar protein concentrations. This method, called the protein probe technique, is based on a peptide conjugated with Eu3+ chelate, and it has already been applied to monitor protein structural changes and small molecule interactions at elevated temperatures. Here, the applicability of the protein probe technique was demonstrated by monitoring single-protein pairing and multiprotein complexes at room and elevated temperatures. The concept functionality was proven by using both artificial and multiple natural protein pairs, such as KRAS and eIF4A together with their binding partners, and C-reactive protein in a complex with its antibody.

中文翻译:

使用无标签蛋白质探针技术的纳摩尔蛋白-蛋白质相互作用监测

蛋白质间相互作用(PPI)是正确细胞功能的重要组成部分,使其成为越来越有趣的药物靶标。传统上,基于Förster共振能量转移的方法已广泛用于PPI研究,但无标记技术最近引起了广泛关注。这些方法是研究PPI的理想选择,最重要的是,无需标记任何一个交互伙伴,从而减少了潜在的干扰和总成本。已经有几种不同的无标记方法可供使用,例如差示扫描量热法和表面等离振子共振,但是这些生物物理方法的通量低至中等,这降低了对PPI抑制剂的高通量筛选(HTS)的适用性。差示扫描荧光法,利用外部荧光探针,是HTS兼容技术,但是实验需要高蛋白浓度。为了改进当前的概念,我们开发了一种基于时间分辨发光的方法,即使在低纳摩尔蛋白浓度下也可以进行PPI监测。这种方法称为蛋白质探针技术,基于与Eu偶联的肽3+螯合物,它已被用于监测高温下蛋白质的结构变化和小分子相互作用。在这里,蛋白质探针技术的适用性通过在室温和高温下监测单蛋白配对和多蛋白复合物来证明。通过同时使用人工和多个天然蛋白质对(例如KRAS和eIF4A及其结合伴侣)以及与抗体形成复合物的C反应蛋白,证明了概念功能。
更新日期:2020-12-15
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