# Molecular Mechanisms of Membrane Transport

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 2020 · $328,466

## Abstract

Project Summary
Active membrane transport is a critical process for normal cell metabolism, including the
maintenance of ion-gradients, osmotic balance, action potentials and apoptosis. The proposed
work will address key questions regarding the mechanisms of nutrient uptake in Escherichia
coli, and it will address questions regarding the structure and organization of these proteins in
the bacterial outer membrane. In E. coli, rare nutrients are sequestered by specific outer-
membrane proteins that derive energy by coupling to the inner-membrane protein TonB. These
TonB-dependent transporters include BtuB, which is responsible for vitamin B12 transport, and
FhuA, FecA and FepA, which are responsible for the transport of various forms of chelated
iron. TonB-dependent transporters, such as BtuB, also acts as receptors for antibacterial
proteins called colicins, which are produced by bacteria to eliminate other bacteria. TonB-
dependent transporters are abundant in Gram negative bacteria and are critical to the proper
functioning of the human microbiome as well as the success of many bacterial pathogens,
such as those that result in meningitis, cholera, pertussis and dysentery. Because they are
unique to bacteria, these transporters are a rational target for the development of new classes
of antibiotics.
High-resolution crystallographic models have been obtained for a number of TonB-dependent
transporters; however, the mechanism by which transport takes place is unclear. The proposed
work will test models for the molecular mechanisms of transport primarily through the use of
site-directed spin labeling and EPR spectroscopy. New approaches have been developed to
perform double electron-electron resonance in intact E. coli, and these approaches will be used
to determine the conformation of TonB-dependent transporters in E. coli under conditions where
transport takes place. In the outer membrane, proteins are sequestered into domains or
islands, which are thought to drive the turnover of outer-membrane proteins in bacteria. EPR
will be used in E. coli to characterize the protein-protein interactions that drive domain formation
and define the supramolecular structure of the outer membrane. Finally, EPR on actively
metabolizing E. coli will be used to test models for the import of colicin E3 into the bacterial cell.

## Key facts

- **NIH application ID:** 9896828
- **Project number:** 5R01GM035215-30
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** DAVID S CAFISO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $328,466
- **Award type:** 5
- **Project period:** 1985-09-06 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9896828

## Citation

> US National Institutes of Health, RePORTER application 9896828, Molecular Mechanisms of Membrane Transport (5R01GM035215-30). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9896828. Licensed CC0.

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