The Behavior of Spin-Labeled Mutants of the Escherichia coli Cobalamin Transporter BtuB Probed by EPR Spectroscopy in Native Environments

Author:
Nyenhuis, David, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Advisor:
Cafiso, David, AS-Chemistry, University of Virginia
Abstract:

Gram-negative bacteria rely on active transport proteins on their outer surface for the import of large, scarce nutrients across the outer membrane. These TonB dependent transporters have two main domains, with a cylindrical β-barrel surrounding a globular hatch domain. The hatch domain must undergo substantial conformational changes to facilitate substrate, but the mechanistic details of the transport process are still unknown. Much of the work to date has focused on the study of these proteins in vitro, but the work presented here focuses on the use of CW and pulsed-EPR techniques to study them in native systems. To this end, EPR results are presented for the E. coli cobalamin transporter BtuB in both whole cells and isolated outer membranes. These results demonstrate conformational responses to substrate binding on the extracellular face of BtuB, and potentially substrate independent extension of the N-terminal ton-box sequence on the periplasmic side. Additionally, pulsed-EPR measurements on BtuB in native systems display novel long-distance components that appear to indicate crowding and potentially organization in the outer membrane. Finally, CW EPR spectra for a set of sites across the extracellular face of BtuB are presented, from which substrate dependent conformational shifts can begin to be mapped out in the native environment. These sites may also help to resolve the nature of the protein’s organization on the OM. These EPR results are supported by the presentation of a method for denoising CW EPR spectra through the stationary wavelet transform.

Degree:
PHD (Doctor of Philosophy)
Keywords:
EPR, wavelet, outer membrane, TBDT, BtuB, DEER
Sponsoring Agency:
NIH NIGMS
Language:
English
Rights:
All rights reserved (no additional license for public reuse)
Issued Date:
2019/04/29