Biology subjects

Ozturk, T. N.

Publications and source records attributed to Ozturk, T. N..

4 recordsLinked to original sources

bioRxiv2025

Apolipoprotein interaction induces shape remodeling and lipid phase separation in giant unilamellar vesicles

Apolipoprotein A-I (ApoA-I) - a 243-residue amphipathic protein containing an N-terminal globular domain and a primarily helical C-terminal lipid binding domain - is a principal protein component of high-density lipoprotein (HDL) or "good" cholesterol, which is an essential component of lipid homeostasis in humans. Synthesized in the liver and intestine and excreted in the blood, ApoA-I undergoes complex, cooperative, and dynamic self-assembly with membrane lipids, producing unlipi-dated (or weakly lipidated), nascent discoidal, and mature HDL states. In vitro studies demonstrate that the reconstitution of purified protein and lipids restores this cooperative self-assembly. However, the kinetic pathways by which these mesoscopic, proteolipidic assemblies form remain incompletely understood. Here, we monitor the dynamics of ApoA-I-membrane interactions through real-time monitoring of morphological changes, which ensue when ApoA-I is incubated with minimal giant unilamellar vesicles (GUVs) composed of single phospholipids or phase-separating phospholipid-cholesterol mixtures. Our fluorescence microscopy measurements reveal that the interaction initiates a gross, morphological remodeling of the parent vesicle proceeding through discrete stages involving membrane poration, solute leakage, vesiculation, and lipid-lipid phase separation. Our atomic force microscopy measurements confirm that the outcome includes discoidal nanoparticles. This qualitative phenomenology is robust and fully reproducible for different protein mutants and alleles (WT APOA-1, {Delta}49ApoA-I, ApoE-3, and ApoE-4) and other lipid mixtures (including mixtures containing phosphoserine lipids). Our molecular simulations recapitulate the essential shape changes and further reveal the composition dependence of the interactions. Together, these findings outline key steps in protein-lipid interactions that facilitate the assembly of mesoscopic reconstituted lipoproteins and nanodiscs.

biophysics↗

bioRxiv2023

Mitigation of membrane morphology defects explain stability and orientational specificity of CLC dimers

Most membrane proteins are oligomers, but the physical forces explaining the stable association of these complexes inside the lipid bilayer are not well understood. The homodimeric antiporter CLC-ec1 highlights the puzzling nature of this reaction. This complex is thermodynamically stable even though it associates via a large hydrophobic protein-protein interface that appears well adapted to interact with the membrane interior. In a previous study, however, we discovered that when CLC-ec1 is dissociated, this interface introduces a morphological defect in the surrounding membrane, leading us to hypothesize association is driven by the elimination of this defect upon dimerization. This study tests this hypothetical mechanism directly and shows it is supported by molecular and physical models. First, using coarse-grained umbrella-sampling molecular simulations, we calculated the membrane contribution to the potential-of-mean-force for dimerization in a POPC bilayer. This shows the stable association of CLC subunits prior to formation of direct protein-protein contacts, but only via the native interface that presents the membrane defect, and not others. Single-molecule photobleaching experiments show that addition of short-chain DLPC lipids, known to alleviate the membrane defect, also shifts the association equilibrium from dimers to monomers. We explain this destabilizing effect through additional umbrella-sampling and alchemical free-energy simulations, which show DLPC enrichment of the defect diminishes the membrane contribution to the association free energy, as it improves the lipid-solvation energetics of the monomer but not the dimer. In summary, this study establishes a physical model that explains the stability and orientational specificity of CLC dimers in terms of membrane-mediated forces, rather than protein-protein interactions. We posit that cells might ubiquitously leverage morphological defects in the bilayer to drive organization of membrane proteins into functional complexes, and that cellular regulation of lipid composition can modulate this organizing effect.

biophysics↗

bioRxiv2020

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

CLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of -10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

biophysics↗

bioRxiv2020

Dimerization of a membrane transporter is driven by differential energetics of lipid solvation of dissociated and associated states

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 caused by 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 proteins 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. IMPACT STATEMENTDifferences in the lipid solvation energetics of associated and dissociated states is a primary driving force for membrane protein oligomerization, presenting a molecular mechanism for lipid regulation in biology.

biophysics↗