A thermodynamic analysis of CLC transporter dimerization in lipid bilayers

Published

October 10, 2023

Author(s)

Rahul Chadda, Taeho Lee, Robyn Mahoney-Kruszka, Elizabeth Kelley, Nathan Bernhardt, Priyanka Sandal, Janice Robertson

Abstract

The CLC-ec1 chloride/proton antiporter is a membrane-embedded homodimer with subunits that can dissociate and associate, but the thermodynamic driving forces favor the assembled dimer at biological densities. Yet, the physical reasons for this stability are confounding as dimerization occurs via the burial of hydrophobic interfaces away from the lipid solvent. For binding of nonpolar surfaces in aqueous solution, the driving force is often attributed to the hydrophobic effect, but this should not apply in the membrane since there is very little water. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG°. To ensure that the reaction reached equilibrium at different temperatures, we utilized a Förster resonance energy transfer assay to report on relaxation kinetics of subunit exchange as a function of temperature. Equilibration times were then applied to measure CLC-ec1 dimerization isotherms at different temperatures using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in Escherichia coli–like membranes exhibits a nonlinear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects such as the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the nonbilayer defect required to solvate the monomeric state is one source of the observed change in heat capacity and indicates the existence of a generalizable driving force for protein association in membranes.

Citation

Proceedings of the National Academy of Sciences of the United States of America-Physical Sciences

Volume

120

Issue

41

Pub Type

Journals

Download Paper

https://doi.org/10.1073/pnas.2305100120

Local Download

Keywords

membrane protein, CLC transporter, dimerization, thermodynamics, lipid membrane

Citation

Chadda, R. , Lee, T. , Mahoney-Kruszka, R. , Kelley, E. , Bernhardt, N. , Sandal, P. and Robertson, J. (2023), A thermodynamic analysis of CLC transporter dimerization in lipid bilayers, Proceedings of the National Academy of Sciences of the United States of America-Physical Sciences, [online], https://doi.org/10.1073/pnas.2305100120, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=956458 (Accessed November 21, 2024)