Fahie Monifa A, Candido Jonathan, Andree Gisele, Chen Min
Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States.
Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States.
ACS Sens. 2021 Mar 26;6(3):1286-1294. doi: 10.1021/acssensors.0c02580. Epub 2021 Feb 18.
Nanopore sensors capable of distinguishing homologous protein analytes are highly desirable tools for proteomics research and disease diagnostics. Recently, an engineered outer membrane protein G (OmpG) nanopore with a high-affinity ligand attached to a gating loop 6 showed specificity for distinguishing homologous proteins in complex mixtures. Here, we report the development of OmpG nanopores with the other six loops used as the anchoring point to host an affinity ligand for protein sensing. We investigated how the analyte binding to the affinity ligand located at different loops affects the detection sensitivity, selectivity, and specificity. Our results reveal that analytes weakly attracted to the OmpG nanopore surface are only detectable when the ligand is tethered to loop 6. In contrast, protein analytes that form a strong interaction with the OmpG surface via electrostatic attractions are distinguishable by all seven OmpG nanopore constructs. In addition, the same analyte can generate distinct binding signals with different OmpG nanopore constructs. The ability to exploit all seven OmpG loops will aid the design of a new generation of OmpG sensors with increased sensitivity, selectivity, and specificity for biomarker sensing.
能够区分同源蛋白质分析物的纳米孔传感器是蛋白质组学研究和疾病诊断中非常理想的工具。最近，一种经过工程改造的外膜蛋白G（OmpG）纳米孔，其门控环6上连接了高亲和力配体，在区分复杂混合物中的同源蛋白质方面表现出特异性。在此，我们报告了以其他六个环作为锚定点来承载用于蛋白质传感的亲和配体的OmpG纳米孔的开发。我们研究了分析物与位于不同环上的亲和配体结合如何影响检测灵敏度、选择性和特异性。我们的结果表明，只有当配体连接到环6时，弱吸附在OmpG纳米孔表面的分析物才能被检测到。相比之下，通过静电吸引与OmpG表面形成强相互作用的蛋白质分析物可被所有七种OmpG纳米孔构建体区分。此外，同一分析物与不同的OmpG纳米孔构建体可产生不同的结合信号。利用所有七个OmpG环的能力将有助于设计新一代对生物标志物传感具有更高灵敏度、选择性和特异性的OmpG传感器。
