Fig. 2: HDX-MS analysis of PLIN3 membrane binding.

a Absolute percentage of deuterium incorporation after 3 sec deuterium exposure of PLIN3 at 1 °C in the absence of liposomes. Each point represents a single peptide, with them being graphed on the x-axis according to their central residue. A domain architecture of human PLIN3 was drawn to match the scale of x-axis of HDX-MS. N-terminal PAT domain (mustard) and 11-mer repeats (cyan) have no detectable secondary structure in the absence of liposomes. The α/β domain and 4-helix bundle are represented in purple and pale pink, respectively. b A predicted 3D structure of human PLIN3 (UniProt: O60664) by AlphaFold (Left). The surface of PAT domain and 11-mer repeats were shown according to the hydrophobicity (Right). The level of hydrophobicity is presented as a color gradient from red (hydrophobic) to gray (polar). c SDS-PAGE and d quantitative analysis of PAT/11-mer repeats and 4-helix bundle liposome binding to DO and 4ME-liposomes. Data are presented as mean values +/− SD from three independent experiments (n = 3). Lane M represents protein ladder. e–g Quantitative analysis of deuterium exchange differences of human PLIN3 in the presence of liposomes. The sum of the difference in the # of incorporated deuterons is shown for the absence and the presence of liposomes over all timepoints. The N-terminus composed of PAT/11-mer repeats were significantly protected from deuterium exchange (defined as > 5% change in exchange, > 0.4 Da mass difference in exchange, a p-value <0.01 using a two-tailed Student’s t-test). Each point represents an individual peptide, with those colored in red having a significant difference, with error bars showing standard deviation (n = 3). Liposomes were generated with e 60 mol% 4ME-PC, 20 mol% 4ME-PE and 20 mol% 4ME-PA, f 80 mol% 4ME-PC, 20 mol% 4ME-PE or g 60 mol% DOPC, 20 mol% DOPE and 20 mol% DAG. A map of deuterium exchange rate according to all peptides throughout the entire PLIN3 was generated based on the AlphaFold predicted PLIN3 structure and color-coded according to the legend. Source data are provided as a Source Data file.