# Protein sequence determinants and properties of the lipid bilayer that govern membrane protein dynamic organization

> **NIH NIH R01** · UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON · 2020 · $423,238

## Abstract

A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and
orients in a lipid bilayer. At least 10% of pathogenic mutations in membrane proteins (MPs) result in mis-
orientation of their transmembrane domains (TMDs). Most studies focus on the protein and the membrane
insertion machinery with little consideration of how lipid environment affects TMD organization. The long-term
goal of this proposal is to understand the role of lipid-protein interactions in the assembly, structure and
function of MPs. Using a combined molecular genetic and biochemical approach, we established that lipid-
dependent TMD orientation is dynamic during and after MP assembly in vivo and is independent of other
cellular factors in vitro. Dependence of TMD topology solely on the intrinsic properties of a MP and its lipid
environment indicates a thermodynamically driven process that can occur in any cell membrane at any time.
We developed a set of lipid mutants of Escherichia coli in which lipid composition can be regulated during or
temporally after MP insertion. This mutant set and a new proteoliposome system in which lipid composition can
be controlled before and after MP reconstitution will be used to test kinetic and thermodynamic limits of TMD
interconversions and establish direct lipid-protein interactions as the basis for in vivo observed phenotypes.
Combining studies of MP and lipid properties led to our formulation of the Charge Balance Rule, which
postulates that charge interactions between lipid head groups and MP extramembrane domains (EMDs) are a
determinant of MP topology. This rule explains stable, dynamic and dual topological organization of a MP and
provides a proof of principle for lipid-dependent assembly of MPs in more complex eukaryotic systems. In Aim1
we will determine the limits imposed on lipid-induced post-assembly changes in MP topology by post-
translation glycosylation and phosphorylation of EMDs. We will determine whether the rate of phosphorylation-
triggered topological changes occur rapidly enough to represent a novel mechanism for metabolic regulation
linked to MP dynamic organization. We will extend our studies to FtsK whose phosphorylation appears to alter
TMD topology as a regulatory mechanism during cell division of E. coli. In Aim 2 we will build biologically based
membrane insertion scales for individual amino acids that incorporate lipid composition, which has not been
previously considered. Understanding how lipid composition affects TMD insertion and orientation will provide
a molecular basis for initial TMD orientation, reversible post-assembly TMD reorientation, MP topological
heterogeneity, and misfolding of mutant or heterologously expressed MPs. In Aim 3 we will analyze how lipid
composition, lipid transbilayer asymmetry and membrane bioenergetic parameters modulate the effective net
charge of EMDs, which will provide an understanding at the mechanistic level of the Charge Balanc...

## Key facts

- **NIH application ID:** 9983711
- **Project number:** 5R01GM121493-04
- **Recipient organization:** UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
- **Principal Investigator:** WILLIAM DOWHAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $423,238
- **Award type:** 5
- **Project period:** 2017-09-15 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9983711

## Citation

> US National Institutes of Health, RePORTER application 9983711, Protein sequence determinants and properties of the lipid bilayer that govern membrane protein dynamic organization (5R01GM121493-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9983711. Licensed CC0.

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