Developing a system to study proteins clustering at membranes

Abstract

Synaptic transmission relies on a dense network of proteins at the postsynaptic density (PSD), where scaffold proteins like PSD‑95 organize neurotransmitter receptors and adhesion molecules into functional complexes. Understanding how these proteins cluster on membranes is key to deciphering synaptic strength and plasticity, yet it remains challenging to study. In living cells, cytoplasmic crowding and limited resolution obscure nanoscale assembly, and past work on PSD‑95 palmitoylation has relied on static, non‑specific interventions or trafficking‑deficient mutants, leaving the real‑time role of membrane anchoring unclear. This work set out to supply the purified, assay‑ready proteins needed for a two‑dimensional membrane reconstitution system that can reveal the step‑by‑step assembly of PSD complexes, laying the groundwork for clearer mechanistic insights and, for strategies to correct synaptic‑organization disorders. To that end, we produced PSD‑95, GluN2B CTD2, and neuroligin CTD with assay‑ready tags. PSD‑95 was made His‑tag‑free after purification; anion‑exchange removed DNA contamination, although low solubility persisted, indicating that future plasmid redesign (for example, adding a solubility tag) may be needed. For GluN2B CTD2, expression in Rosetta2 E. coli and the inclusion of 2 M urea throughout purification increased the soluble yield and markedly reduced aggregation at pH 7.4. Neuroligin CTD experienced a greater than 90 % loss somewhere in the fluorescent‑labeling workflow; it’s extremely low 280 nm absorbance makes this difficult to track. To enable precise monitoring, additional His‑tagged batches were prepared in BL21(DE3), and future expression in Rosetta2 is proposed to offset rare‑codon limitations and improve output. Despite remaining hurdles, these optimized protein preparations provide the essential building blocks for forthcoming membrane‑based clustering assays, bringing us closer to observing how PSD proteins self‑assemble and remodel in real time.