The central enigma of protein folding lies in how the physical forces of nature drive a simple string of amino acids into a stable, conformationally defined protein. 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. Preliminary studies show that the protein can be shifted back to the dimer state with additional mutations or in certain lipid conditions. These results present CLC-ec1 as a model for the study of reversible dimerization, which simplifies the protein folding process while still encompassing all of the thermodynamic properties of protein interactions in the membrane environment. To make these energetic measurements, the monomer/dimer populations will be quantified using three well-established methods: (i) Poisson-counting of monomer vs. dimers in liposome populations, (ii) fluorescence self-quenching in liposomes, and (iii) Frster resonance energy transfer (FRET) in liposomes and supported bilayers for single molecule studies. With these assays in place, experiments will be carried out to investigate two alternative hypotheses that have pervaded discourse in this field. First, that specific transmembrane helix interactions are 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, with significant positions studied further by increasing side- chain volume to modulate the van der Waals interactions. The second hypothesis is that interactions are driven by increased entropy of lipids upon helix association. To study this, the molecules forming the lipid solvent will be modified by changing the chemical head group, chain length and chain order using unsaturated or tetra-ether lipids from archaea. For all experiments, free energy relationships will also be measured with respect to temperature to extrapolate values for enthalpy and entropy. These results will provide insight into the driving forces for membrane protein interactions, and may even provide a foundation for attacking general questions underlying protein folding in the strange solvent that is the lipid bilayer.

Public Health Relevance

Membrane proteins are molecular 'gate-keepers' regulating the passage of biological materials across the lipid bilayer. As such, they are critically involved in physiological processes and may be key therapeutic targets. By understanding the energetic factors governing how these proteins interact and assemble in the lipid environment, we will gain insight into methods of modulating membrane protein function and cell physiology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Transition Award (R00)
Project #
5R00GM101016-05
Application #
8901201
Study Section
Special Emphasis Panel (NSS)
Program Officer
Chin, Jean
Project Start
2012-06-01
Project End
2016-07-31
Budget Start
2015-08-01
Budget End
2016-07-31
Support Year
5
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Iowa
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52246
Chadda, Rahul; Cliff, Lucy; Brimberry, Marley et al. (2018) A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers. J Gen Physiol 150:355-365
Leisle, Lilia; Chadda, Rahul; Lueck, John D et al. (2016) Cellular encoding of Cy dyes for single-molecule imaging. Elife 5:
Chadda, Rahul; Krishnamani, Venkatramanan; Mersch, Kacey et al. (2016) The dimerization equilibrium of a ClC Cl(-)/H(+) antiporter in lipid bilayers. Elife 5:
Chadda, R; Robertson, J L (2016) Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy. Methods Enzymol 581:53-82
Stockbridge, Randy B; Robertson, Janice L; Kolmakova-Partensky, Ludmila et al. (2013) A family of fluoride-specific ion channels with dual-topology architecture. Elife 2:e01084