# Membrane Protein Folding

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2021 · $305,809

## Abstract

PROJECT SUMMARY
The ClC family of chloride channels and transporters play important physiological roles. Mutations in ClC
family proteins are associated with an array of diseases, some of which are caused by protein misfolding.
Thus, it is important to understand how these protein fold. Yet the folding of ClC family proteins presents a
formidable challenge, for both biology and experiment. Not only are they large membrane proteins, but they
have a remarkably complex topology with many helices that insert only part way into the bilayer and then loop
back out, leaving considerable polar surfaces in the center of the bilayer. How proteins like this can fold in the
apolar bilayer remains unknown and largely unexamined.
Using a new single molecule forced unfolding technique we developed in the prior grant period, we made some
surprising discoveries regarding the folding of the ClC antiporter from E. coli that we now propose to
investigate more deeply. In particular, it appears that the protein folds in two domains that would require
plunging polar moieties into the apolar bilayer. Why would the protein be designed to fold in this manner and
how might it be accomplished? To address these issues we propose the following aims:
Aim I: Structure of isolated ClC domains. We will investigate the structures of the domains with polar exposed
surfaces in isolation and how they adapt to the bilayer environment.
Aim II: How do lipid properties affect the stability of the isolated domains? To gain insight into the interaction
of the protein and lipids during unfolding and folding, we will examine the effects of different lipids on the
energetics of domain separation.
Aim III: Can separate folding of the domains increase folding efficiency? Is there evidence for N- to C-terminal
folding? We hypothesize that dividing the folding of ClC-ec into two units simplifies the folding of such a large
complex protein. As the protein emerges from the translocon, the N-terminal domain may commence folding,
reducing options for the C-terminal domain and possibly templating C-terminal domain folding. To test this
hypothesis we will examine the folding efficiency and folding kinetics of the individual domains by themselves
and in the presence of the other domain.
Our work will map the folding energetics and pathway for arguably the most complex protein ever studied at
this level; provide insight into the folding of the large number of membrane proteins with re-entrant helices; and
provide new ideas for intervening in membrane protein folding diseases.

## Key facts

- **NIH application ID:** 10241960
- **Project number:** 5R01GM063919-20
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** JAMES U BOWIE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $305,809
- **Award type:** 5
- **Project period:** 2001-08-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10241960

## Citation

> US National Institutes of Health, RePORTER application 10241960, Membrane Protein Folding (5R01GM063919-20). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10241960. Licensed CC0.

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