# Structural and functional analysis of novel microbial membrane proteins

> **NIH NIH R35** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2024 · $432,825

## Abstract

Microorganisms contend with a host of environmental threats, ranging from chemical toxins to dynamically
changing ionic conditions. In response, microbes have evolved a unique catalog of membrane transporters and
signaling proteins. My research program integrates cutting edge approaches in electrophysiology, membrane
protein biophysics, x-ray crystallography, and cryo-EM for the molecular and physiological characterization of
membrane proteins that contribute to uniquely microbial physiologies. Currently, three major areas of inquiry
are 1) molecular mechanisms for membrane export of environmental toxins 2) molecular mechanisms of
receptors that sense and integrate information about changing ionic gradients 3) development of new
approaches to overcome challenges in structural characterization of small membrane proteins. For the first line
of inquiry, we build off our identification and characterization of two previously unannotated microbial
physiologies, fluoride and guanidinium export. These ions are common in the microbial milieu and have broad-
spectrum inhibitory effects on microbial metabolism. We provided the first identification and mechanistic and
structural characterization of bacterial exporters of these toxins. Future efforts will focus on a) determining the
molecular mechanism and first structure of fluoride exporters of pathogenic eukaryotic microbes, known as
FEX. These studies will provide molecular information that can be applied to inhibitor design, as well as broad-
based insight into membrane protein evolution. b) biophysical analysis of guanidinium exporter Gdx. Together
with our recent structures, this project will reveal the mechanistic basis for promiscuous substrate recognition
and substrate-coupled conformational change, generating key insight into multidrug transport mechanisms
more generally. For the second line of research, we will establish molecular mechanisms of signaling proteins
that enact biofilm or virulence programs in response to changing ionic conditions. Our first target is a histidine
kinase receptor, KinC, that detects changes in environmental potassium. To understand the biophysical basis
for receptor activation, we will evaluate structural ensembles by cryoEM, employ site-directed mutagenesis and
in vivo assays for receptor activation, and reconstitute signaling function in lipid vesicles. This work will pioneer
biophysical research into ion-gradient-responsive signaling, with implications for pathogenic processes like
host colonization and biofilm growth. Our third major research thrust is to develop new approaches to
overcome challenges of structural characterization of small membrane proteins. We recently designed a new
and efficient approach to generate crystallization chaperones and cryo-EM fiducials, and we will continue to
develop this technology in order to make it accessible for as many membrane protein targets and labs as
possible. Together, these research activities will generate novel insigh...

## Key facts

- **NIH application ID:** 10865014
- **Project number:** 5R35GM128768-07
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Randy B. Stockbridge
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $432,825
- **Award type:** 5
- **Project period:** 2018-08-01 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10865014

## Citation

> US National Institutes of Health, RePORTER application 10865014, Structural and functional analysis of novel microbial membrane proteins (5R35GM128768-07). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10865014. Licensed CC0.

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