Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.

Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.

Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

2019

Month Degree Awarded

February

First Advisor

Min Chen

Subject Categories

Biochemistry | Molecular Biology | Other Biochemistry, Biophysics, and Structural Biology

Abstract

Pore forming proteins (PFPs) are membrane channels that are essential for various biological processes. For example, some PFPs act as gatekeepers of the cell, controlling the traffic of ions and macromolecules flowing into and out of cells; while others are involved in causing cell death (Reiner et al., 2012). Our fundamental understanding of PFPs determines our ability to employ these proteins for use in biomedical research and nanopore technology. Given their nanoscale dimensions, reproducibility and functionality these PFPs are widely used in the growing field of nanopore technology, particularly nanopore sensing (Reiner et al., 2012; Feng et al., 2015). These biological nanopores are powerful tools enabling the production of a real-time, sensitive and selective technology. Ultimately, we aim to enhance our current understanding of PFPs to improve the currently available nanopore sensing technology.

In this work, we report a nontraditional biological nanopore, Outer membrane protein G (OmpG) and its ability to detect protein. The principle of detection for this biosensor is harnessed via the dynamic movement of loop 6. Through chemically modifying the pore we attached a ligand to search or fish for our target protein of interest. In addition to successful protein detection, we were also able to discriminate between homologous within an antibody mixture. Moreover, the sensitivity of our sensor discloses a unique fingerprint for each protein target detected. This result led us to further investigate the loop network of OmpG which is vital to our detection system. The OmpG pore is pH-sensitive, pH influences the conformation of the pore. We hypothesized that the loop network of OmpG can be influenced by charge, allowing us to manipulate the pore conformation independent of pH. We report a pH-insensitive pore that allows us to tailor OmpG for sensing an array of protein analytes. Also included in this work, we investigate protein and peptide detection and translocation by traditional nanopores to build on our research interest.

Available for download on Saturday, February 01, 2020

Share

COinS