Neutral lipid triglycerides a main reserve for fat and energy are stored in organelles called lipid droplets. NMR study around the PLIN1 in combination with GSK2636771 molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a systems have recently become available for structural and functional studies. Recombinant PLIN1 has been purified and reconstituted in lipid droplet-like particles [13 14 Using an system it was shown that phosphorylation of PLIN1 enhances the triglyceride lipase activity demonstrating the direct connection between PLIN1 phosphorylation and the activation of lipolysis [13]. Hypothetically PLIN1 phosphorylation causes changes around the droplet surface making the internal triglycerides more accessible to the GSK2636771 lipase [11]. The ability to reconstitute PLIN1 in lipoprotein particles opens the possibility to design structural studies to advance our understanding of the mechanisms of lipolysis regulation. Nevertheless these lipoprotein particles are too large (~20 nm diameter) for solution NMR studies and they are very difficult if not impossible to form diffraction quality single crystals for crystallography studies. Fortunately several other structural techniques could be GSK2636771 applied to this type of samples. For example topologies of lipoprotein complexes have been decided using solid-state NMR [15 16 fluorescence spectroscopy [15-18] and electron paramagnetic resonance (EPR) [19]. Among these solid-state NMR is particularly suitable to study these lipoprotein complexes because three-dimensional structure details could be obtained [22-29]. Here we report both NMR experimental data and structural models that could be useful to advance our understanding of protein targeting to lipid droplet and the function of PLIN1. 2 Material and methods 2.1 Protein expression and purification Isotopically enriched (13C 15 ISOGRO 15 and uniformly labeled 13C-glucose were purchased from Sigma-Aldrich (St. Louis MO). Lipid 1 2 (DMPG) was purchased from Avanti Polar Lipids (Alabaster AL). Benzonase was purchased from EMD Millipore (Billerica MA). The over-expression of PLIN1 (CG10374 “type”:”entrez-protein” attrs :”text”:”NP_732904.2″ term_id :”45551956″ term_text :”NP_732904.2″NP_732904.2) as a fusion protein with thioredoxin-[His]6-Stag was carried out as previously reported [13] with slight modifications to incorporate stable isotopes for NMR studies. Transformed Rosetta cells with the recombinant plasmid (pET32-CG10374) were produced in 200 mL Luria broth medium at 37 °C until optical density 0.8 at 600 nm. The bacteria pellet was collected by centrifugation and cultured in 1 liter M9 minimal medium made up of reagents enriched with NMR-active stable isotopes (13C-glucose and 15NH4Cl). The medium was supplemented with properly labeled algae extracts (ISOGRO) to boost IL10 protein yield. When optical density reached 0.8 protein expression was induced by addition of GSK2636771 1 1 mM IPTG. After 6 hours cells were harvested by centrifugation. Thioredoxin-Lsd1 fusion protein was GSK2636771 purified essentially as previously described [12]. The final protein pellet was resuspended in 20 mM Tris pH 8.0 6 M Urea 150 mM NaCl 10 mM dithiothreitol and a solution of protein stock (1.7 mg/mL) was stored in the freezer. 2.2 Reconstitution of thioredoxin-PLIN1 in lipoprotein particles and thrombin cleavage Thioredoxin-PLIN1/DMPG complexes were GSK2636771 prepared as previously described with a final lipid to protein ratio of 70:1 [12]. After exhaustive dialysis thioredoxin-PLIN1/DMPG complexes were brought to 60% (w/v) sucrose and subjected to ultracentrifugation in a sucrose density gradient (30 to 60 %60 % (w/v)). The distinct white band floating at a density of 1 1.17g/ml was collected as a single.