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首页> 外文期刊>Chemistry: A European journal >A Versatile Polypeptide Platform for Integrated Recognition and Reporting:Affinity Arrays for Protein-Ligand Interaction Analysis
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A Versatile Polypeptide Platform for Integrated Recognition and Reporting:Affinity Arrays for Protein-Ligand Interaction Analysis

机译：用于集成识别和报告的多功能多肽平台：用于蛋白质-配体相互作用分析的亲和力阵列

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摘要

A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding.The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues.The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration.An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold.The affinities of the array members for human carbonic anhydrase II (HCAII)were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant K_d=0.02-3 muM).The affinity of HCAII for acetazol-amide (K_d=4 nM)was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders.Also,successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling.Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry,diagnostics and pharmaceutical chemistry.
机译：报道了一个蛋白质检测和定量的分子平台，该平台已将识别与直接监测靶蛋白的结合整合在一起。该平台基于多用途的42个残基的螺旋-环-螺旋多肽，该多肽二聚化形成四螺旋束并允许在单个可寻址赖氨酸残基的侧链上进行识别和报告元件的位点选择性修饰。模型目标蛋白碳酸酐酶及其抑制剂苯磺酰胺之间的良好相互作用被用于概念验证。设计阵列，将具有脂肪族或低聚甘氨酸间隔基的苯磺酰胺衍生物和荧光丹磺酰报告基团引入支架中。通过用目标蛋白滴定确定阵列成员对人碳酸酐酶II（HCAII）的亲和力，发现受隔离物的特性高度影响（解离常数K_d = 0.02-3μM）。在与苯磺酰胺阵列成员之一的竞争实验中确定了HCAII对乙酰唑酰胺（K_d = 4 nM）的亲和力，以解决为新的靶蛋白结合剂筛选物质库的可能性。不同碳酸酐酶同工酶之间成功的亲和力区分突显了进行未来亚型表达谱分析的可能性。我们的平台预计将成为生物化学，诊断学和药物化学中各种生物传感器和蛋白质微阵列应用的灵活工具。

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