A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers

Chadda, Rahul; Cliff, Lucy; Brimberry, Marley; Robertson, Janice

doi:10.1085/jgp.201711893

Cited by 21 publications

(50 citation statements)

References 27 publications

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“…These membranes are a synthetic mimic of the E. coli polar-lipid composition, containing C 16:0,18:1 tails, the most common acyl chain in biological membranes (Phillips, 2018). While CLC-ec1 forms homodimers in detergent and membranes (Maduke et al, 1999; Dutzler et al, 2002), our measurements revealed this protein complex results from association of two functionally competent monomers (Robertson et al, 2010) via a membrane-embedded hydrophobic interface (Figure 1B), with an equilibrium dimerization free energy of −10.9 kcal/mole, relative to a standard state of 1 monomer/lipid (Chadda et al, 2018). This is a remarkable finding, implying that the population of dissociated monomers in biological conditions is virtually zero.…”

Section: Introductionmentioning

confidence: 75%

“…DNA constructs for CLC-ec1 C85A/H234C (WT), C85A/H234C/I201W/I422W (WW) (Chadda et al, 2016) and C85A/H234C/R230C/L249C (RCLC) were described previously (Chadda et al, 2018). Expression and purification of these CLC-ec1 variants was carried out as described earlier (Chadda et al, 2016).…”

Section: Methodsmentioning

confidence: 99%

“…Proteoliposomes samples were extruded, imaged on TIRF microscope, and the videos analyzed for counting single-molecule photobleaching steps as described earlier (Chadda et al, 2016; Chadda and Robertson, 2016; Chadda et al, 2018). Briefly, dialyzed proteoliposomes were freeze-thawed seven times leading to formation of large multilamellar vesicles (MLVs).…”

Section: Methodsmentioning

confidence: 99%

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Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states

Chadda¹,

Bernhardt

Kelley

et al. 2020

Preprint

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Over two-thirds of membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins in the membrane remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid an energetic penalty for solvating a thinned-membrane defect caused by their exposed dimerization interfaces. Supporting this theory, we demonstrate that this penalty is drastically reduced with minimal amounts of short-chain lipids, which stabilize the monomeric state by preferentially solvating the defect rather than altering the physical state of the membrane. We thus posit that the energy differentials for local lipid-solvation define membrane-protein association equilibria, and describe a molecular-level physical mechanism for lipid regulation of such processes in biological conditions.

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Section: Introductionmentioning

confidence: 75%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states

Chadda¹,

Bernhardt

Kelley

et al. 2020

Preprint

Self Cite

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show abstract

“…1B). Specifically, the measured equilibrium dimerization free energy for this complex is -10.9 kcal/mole, relative to a standard state of 1 monomer/lipid (Chadda et al, 2018). This is a remarkable finding, in that it implies that the population of dissociated monomers at biological proteinexpression levels is virtually zero.…”

Section: Introductionmentioning

confidence: 81%

“…4D,E) demonstrates no changes in the quality of images obtained in the different lipid conditions. While the cryo-EM imaging indicated no significant differences in liposome size distributions, we also examined the photobleaching probability distributions of two experimental controls: I201W/I422W, referred to as 'WW', a version of the protein with two tryptophan substitutions at the dimerization interface (Robertson et al, 2010) that reports the fixed monomer probability distribution; and R230C/L249C, or 'RCLC', a disulfide crosslinked constitutive dimer (Nguitragool and Miller, 2007) that reports the fixed dimer probability distribution (Chadda et al, 2018). Photobleaching analysis of these controls in 20% DL liposomes show dependencies on the protein mole fraction comparable to those observed for the 2:1 POPE/POPG composition (Fig.…”

Section: Short-chain Lipids Shift Clc Dimerization Equilibrium Without Effect On Protein Function or Global Membrane Changesmentioning

confidence: 99%

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states

Chadda

Bernhardt

Kelley

et al. 2021

eLife

Self Cite

View full text Add to dashboard Cite

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium towards the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

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Occupancy distributions of membrane proteins in heterogeneous liposome populations

Cliff¹,

Chadda²,

Robertson³

2020

Biochimica et Biophysica Acta (BBA) - Biomembranes

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A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers

Cited by 21 publications

References 27 publications

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states

Occupancy distributions of membrane proteins in heterogeneous liposome populations

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