The development of a hydrogel-supported phospholipid membrane for the reconstitution of transmembrane proteins
Bibliographic record
Abstract
The overall objective of this work was to make a hydrogel-supported phospholipid bilayer that models a cytoskeleton-supported cell membrane. Hydrophobic anchors attached to a hydrophilic hydrogel surface were proposed to promote the adhesion of liposomes and cause the self-assembly of a phospholipid bilayer membrane on that hydrogel surface. The phospholipid-hydrogel conjugate, called the pre-Lipobead, was composed of a crosslinked poly(dimethylacrylamide) core and surface-grafted phospholipids. Using a one-step radical suspension polymerization scheme and phospholipids in which reactive vinyl groups had been attached, the anchors preferentially located at oil-water interfaces during pre-Lipobead formation and through their vinyl groups, became covalently attached at the hydrogel surface. X-ray photoelectron spectroscopy and laser scanning confocal microscopy confirmed surface localization of the anchors. Upon mixing the pre-Lipobeads with liposomes, a lipid membrane readily self-assembled around the hydrogel surface, forming the Lipobeads. The synthesis scheme allowed control of surface anchor density and thus potential control of membrane properties. The properties of the formed Lipobead membrane were investigated by fluorescence quenching and fluorescence-recovery-after-photobleaching (FRAP). The quenching of fluorescently labeled phospholipids (PC-NBD) in the Lipobead suggested that the formed membrane was mostly a bilayer. The lateral diffusivity of PC-NBD in the membrane, measured by FRAP, was found to vary with the surface anchor and hydrogel crosslinking densities. The Lipobead membrane could form a diffusion barrier to large hydrophilic solutes such as dextran, but permeable to small ions such as Ca2+. The ability of Lipobeads to incorporate transmembrane proteins was investigated by incubating proteoliposomes containing muscarinic m2 acetylcholine receptors with the Lipobeads. Fluorescent antibodies that target an extracellular fragment of the receptor coupled with confocal microscopy revealed the presence of receptors on the Lipobead surface with some proteins reconstituted in the right orientation. Specific binding of an antagonist to those reconstituted receptors indicated that the drug-binding pockets were intact and the reconstituted receptor density was between 132–152 receptors/μm2, a value comparable to receptor density on cardiac cell surfaces. The hydrogel-supported bilayer membrane shows promise as a new tool for studying membrane and transmembrane protein biology, as well as potential applications in high throughput drug screening, diagnostics, and drug delivery.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.001 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".