# Driving forces of membrane protein assembly in membranes

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2020 · $337,842

## Abstract

ABSTRACT
What are the thermodynamic driving forces that influence the free energy of membrane protein folding and
assembly in lipid bilayers? For soluble proteins, the burial of hydrophobic groups away from aqueous
interfaces is a major driving force, but membrane-embedded proteins cannot experience hydrophobic forces,
as the lipid bilayer lacks water. A fundamental conundrum thus arises: how does a greasy protein surface find
its greasy protein partner in the greasy lipid bilayer to fold faithfully into its native structure? Recently, a
structurally stable and functional monomeric form of the normally homodimeric Cl-/H+ antiporter CLC-ec1 was
designed by introducing tryptophan mutations at the dimer interface. We have used this to develop a new
model system for studying reversible dimerization in membranes for free energy measurements, which
simplifies the protein folding process while still encompassing all of the thermodynamic properties of protein
interactions in the membrane environment. To quantify monomer vs. dimer populations across a wide range of
protein per lipid mole ratios, we developed (i) Förster resonance energy transfer (FRET) and (ii) single-
molecule photo bleaching by total internal reflection microscopy in liposomes methods for the CLC-ec1 system.
The sensitivity of single-molecule microscopy allows us to go to extremely dilute conditions where we observe
dissociation of CLC-ec1 in membranes. With measurements of the energetics already in place, we will
investigate two alternative hypotheses that have pervaded discourse in this field. First, that protein association
is enthalpy-driven by van der Waals forces at highly complementary surfaces. Changes in free energy will be
measured upon substitution of interface residues to alanine or tryptophan, and efforts made to identify if VDW
motifs can be conferred to already destabilized constructs. The second hypothesis is that interactions are
driven by increased entropy of lipids upon subunit association. To study this, the molecules forming the lipid
solvent will be modified by testing hydrophobic mismatch as a function of acyl chain length, and also the
depletion-attraction force by changing lipid radius of gyration, e.g. larger unsaturated and tetraether lipids vs.
smaller non-polar general anesthetics. For all experiments, free energy relationships will be measured as a
function of temperature to extrapolate enthalpy and entropy changes. This research will be carried out by a
team of interdisciplinary scientists in the Robertson laboratory, with levels of training from graduate student,
postdoc, research scientist and principal investigator, combining expertise of membrane protein biochemistry,
single-molecule microscopy and computational modeling to provide an unlimited investigation into this
important biophysical question. The results from this study will provide a physical foundation for the
development of informed strategies aimed at correcting protein mis-folding or regula...

## Key facts

- **NIH application ID:** 9985875
- **Project number:** 5R01GM120260-06
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Janice L Robertson
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $337,842
- **Award type:** 5
- **Project period:** 2016-08-01 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9985875

## Citation

> US National Institutes of Health, RePORTER application 9985875, Driving forces of membrane protein assembly in membranes (5R01GM120260-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9985875. Licensed CC0.

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