Our central goal is to create a nanopore sensor that can be tuned to specifically detect virtually any of these disease-related protein. The sensor is an engineered form of outer membrane protein G (OmpG) from E. coli. The loops that connect the strands of OmpG's ?-barrel are either appended with a ligand or lengthened with a recognition sequence to create the specific sensing elements. Our preliminary results demonstrate that the OmpG nanopore sensor is remarkably sensitive, able to distinguish variants within a mixture of antibodies that were all raised against the same hapten. To expand the utility of the sensor, we will explore the fundamental mechanisms that govern sensor-target interactions. Furthermore, the incorporation of new binding sites within OmpG's loops will proceed by two routes. The first is rational design, where we will incorporate known polypeptide sequences that recognize established targets. The second route takes advantage of OmpG's expression in the E. coli outer membrane. A randomized library of OmpG mutants will be selected for novel target affinity directly from the bacteria using a high- throughput screening and enrichment approach.

Public Health Relevance

Detection of protein-based biomarkers is of great interest in the diagnosis and subsequent treatment of cancer or viral infection. This proposal outlines a new approach to create a nanopore sensor platform that can be rapidly tuned to detect virtually any specific protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM115442-04
Application #
9667445
Study Section
Nanotechnology Study Section (NANO)
Program Officer
Wu, Mary Ann
Project Start
2016-04-01
Project End
2021-03-31
Budget Start
2019-04-01
Budget End
2020-03-31
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Massachusetts Amherst
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Hadley
State
MA
Country
United States
Zip Code
01035
Wolfe, Aaron J; Gugel, Jack F; Chen, Min et al. (2018) Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics. J Phys Chem B 122:9471-9481
Perez-Rathke, Alan; Fahie, Monifa A; Chisholm, Christina et al. (2018) Mechanism of OmpG pH-Dependent Gating from Loop Ensemble and Single Channel Studies. J Am Chem Soc 140:1105-1115
Wolfe, Aaron J; Gugel, Jack F; Chen, Min et al. (2018) Detergent Desorption of Membrane Proteins Exhibits Two Kinetic Phases. J Phys Chem Lett 9:1913-1919
Wolfe, Aaron J; Si, Wei; Zhang, Zhengqi et al. (2017) Quantification of Membrane Protein-Detergent Complex Interactions. J Phys Chem B 121:10228-10241
Tang, Ke; Zhang, Jinfeng; Liang, Jie (2017) Distance-Guided Forward and Backward Chain-Growth Monte Carlo Method for Conformational Sampling and Structural Prediction of Antibody CDR-H3 Loops. J Chem Theory Comput 13:380-388
Wolfe, Aaron J; Hsueh, Yi-Ching; Blanden, Adam R et al. (2017) Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy. Anal Chem 89:8013-8020