Fig. 3

LIMP-2 binding to liposomes characterized by SPR, NS-EM, and DLS. a Binding kinetics of LIMP-2 to liposomes at pH 7.5 were characterized by SPR. Representative SPR sensorgrams (colored traces) of LIMP-2 monomer (left) and dimer (right) binding to immobilized POPC (top panel), DOPS:DOPC (3:2) (middle) and POPS liposomes (bottom) at concentrations indicated were overlaid with kinetic fitting curves (black) using 1:1 kinetic model. The binding responses are scaled according to dimer-POPS responses. Immobilized levels of POPC, DOPS:DOPC (3:2), and POPS on the L1 SPR chip were 8900, 7500, and 6000 units, respectively. b Examples of negative stain EM micrographs indicate the LIMP-2 dimer bridges POPS liposomes and induces clustering of liposomes. Red arrow indicates a clear example of two liposomes forming a tight association with a clear boundary bridged by LIMP-2 dimer (insert is enlarged view). Blue arrow indicates a grape bundle shaped cluster of multiple liposomes. These features are not observed in any images of BSA and LIMP-2 monomer samples, which were used as negative controls. Total numbers of images observed were 5, 8, 16, and 12 for BSA + POPS, monomer + POPS, POPS, and POPS + LIMP-2 dimer, from duplicated experiments. c Addition of LIMP-2 dimer into POPS liposome (red) shifts the DLS profiles of POPS liposome (black). POPS liposome with BSA (blue) used as a negative control. The average radii are 50.6 ± 0.4, 6.53 ± 0.06, 68.6 ± 1.1, 3.84 ± 0.02, 52.4 ± 4 Å for PS liposome, LIMP-2 dimer, PS + LIMP-2 dimer, BSA, and PS + BSA, respectively from 15 measurements of each duplicated experiments. The estimated molecular weights of POPS and POPS + LIMP-2 dimer are 32,774 ± 1300 and 66,557 ± 2500 KD, respectively