Effect of Lipidic Cubic Phase Structure on Functionality of the Dopamine 2L Receptor: Implications for in Meso Crystallization

Abstract

The success of the lipidic cubic phase for crystallization, particularly of integral membrane proteins, is increasing. In the past two years, more than 25% of membrane protein structures have been solved within the biomimetic environment of the lipidic cubic phase. However, the relationship between the lipid matrix and crystal growth still remains a mystery. Herein we show that the bilayer structure of the lipidic cubic phase is crucial to retention of the functionality of the dopamine D2 long receptor. Destruction of the cubic architecture at higher protein concentrations is associated with a significant drop in the amount of functional receptor. This has profound implications for in meso crystallization and suggests that preliminary experiments to determine the maximum protein loading within the lipidic cubic phase must be carried out prior to in meso crystallization experiments.

Cubic Liquid Crystalline Nanostructures Involving Catalase and Curcumin: BioSAXS Study and Catalase Peroxidatic Function after Cubosomal Nanoparticle Treatment of Differentiated SH-SY5Y Cells

“…The cubic unit cell dimensions increased in the presence of an associated catalase enzyme, while retaining the overall cubic phase organization. The structural influence of protein incorporation into liquid crystalline lipid assemblies has been discussed in several publications [8,9,11,20,21,22,23,24,25,26,27,28]. For instance, neurotrophin BDNF confinement resulted in the formation of multiphase and multicompartment liquid crystalline lipid nanoparticles [11].…”

“…High encapsulation efficacy has been reported for hydrophilic guest macromolecules such as brain-derived neurotrophic factor (BDNF), ovalbulmin and protein vaccines [11,20,21,22,23,24]. Soluble, peripheral, and integral membrane proteins have been studied in LCPs in relation to in meso protein crystallization, biosensor development involving encapsulated enzymes, and drug-delivery systems [21,22,23,24,25,26,27,28]. LCP-derived nanocarriers are increasingly used in a range of applications employing lipophilic drugs and theranostic agents [14,15,16,17,18,19].…”

Toward Cell Membrane Biomimetic Lipidic Cubic Phases: A High-Throughput Exploration of Lipid Compositional Space

“…Their fundamental bilayer structure also naturally mimics the native cell membrane, making them ideal for the encapsulation of membrane-associated and integral membrane proteins . Due to its biomimetic nature, the lipidic cubic phase can provide an ideal environment for the encapsulation of hydrophilic and hydrophobic proteins, − peptides , and nucleic acids with a range of molecular weights.…”

Direct demonstration of lipid phosphorylation in the lipid bilayer of the biomimetic bicontinuous cubic phase using the confined enzyme lipid A phosphoethanolamine transferase

“…Results obtained under identical conditions after 2 hours were found to be irreproducible, consistent with a previous observation that a time period of several hours is required for full reconstitution of the protein into the cubic phase. 28 Activity of NmEptA in DMPE doped MO. The activity of NmEptA was also investigated in the presence of the saturated chain phospholipid DMPE, where DMPE causes an increase in Q D II lattice parameters from B105 Å to B108 Å at 10% w/w, Fig.…”

“…Ligand binding assays were recently used to show that the dopamine D2L receptor retained a conformation that preserves the ligand binding region when encapsulated in some bicontinuous cubic phases. 28 However, to our knowledge the activity of a large amphiphilic protein, which has amphiphilic donor and receiver substrates, when encapsulated in the bicontinuous cubic phase has not yet been reported. These systems differ in that the hydrophilic domain must be confined within the nanoscale aqueous channel network of the cubic phase.…”