Schulte Clemens, Khayenko Vladimir, Nordblom Noah Frieder, Tippel Franziska, Peck Violetta, Gupta Amit Jean, Maric Hans Michael
Rudolf Virchow Center, Center for Integrative and Translational Bioimaging, University of Wuerzburg, Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany.
Nanotemper Technologies GmbH, Flößergasse 4, 81369 Munich, Germany.
iScience. 2020 Dec 7;24(1):101898. doi: 10.1016/j.isci.2020.101898. eCollection 2021 Jan 22.
Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.
蛋白质-蛋白质相互作用（PPI）对于我们理解生理学和病理学至关重要。涉及短线性基序的PPI在免疫识别、信号传导和调节中起主要作用，并为药物干预提供了有吸引力的切入点。然而，目前最先进的蛋白质-肽亲和力测定方法通量和灵敏度有限，这通常是由于配体固定、标记或合成所致。在此，我们介绍一种利用温度相关强度变化（TRIC）现象对蛋白质-肽相互作用进行溶液内分析的高通量方法。我们使用TRIC来鉴定和精细定位低亲和力和高亲和力蛋白质相互作用位点，并确定序列结合要求。通过使用野生型和突变重组蛋白以及组织裂解物中的天然蛋白进行基于微阵列的研究来实现验证。芯片上的中和作用以及与结构数据的强相关性确立了TRIC作为一种准无标记方法，可用于测定具有大动态范围的未修饰肽库的结合亲和力。
