How We can proceed for the study of NPC1 protein (a lysosomal membrane protein) structural changes?

Reconstitute NPC1 in nanodiscs and compare ±cholesterol/ligands by cryo-EM, then map conformational shifts with HDX-MS or crosslink-MS plus MD simulations.

In cells, build an intramolecular NPC1 FRET biosensor targeted to lysosomes to track state changes under cholesterol flux perturbations (e.g., NPC2 addition, U18666A) and correlate with FRAP/trafficking readouts.

Validate structure–function by cholesterol transport assays and, if needed, smFRET or site-directed spin labeling/EPR on purified NPC1.