Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations

Abstract

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at ?14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

“…The bR Protein Is Inserted within NLP Membrane-bR⅐NLPs from cell-free co-expression and NLPs assembled by conventional means (10,25) were both examined by AFM to assess NLP size and shape and to demonstrate the association between bR with NLPs. For comparison with cell-free produced bR⅐NLP complexes, both bR⅐NLPs and empty NLPs were also prepared using methods described previously (1,10).…”

Kinetics of lipid mixing between bicelles and nanolipoprotein particles

“…Then the bicelle and NLP would separate. NLPs are known to display a distribution of different sizes [40, 41] presumably reflecting different apolipoprotein conformations [8]. In this model, at the reaction end point the apolipoproteins would still be bound to lipids as NLPs, but they would have smaller diameters, as observed [5], presumably due to the greater stability of bicelles compared with NLPs.…”

Measurement of single low-density lipoprotein particles by atomic force microscopy

“…Previous studies have reported AFM-based evaluations of the shapes and sizes of lipoprotein classes such as very low-density lipoproteins (VLDL), 16 LDL, [16][17][18][19] and high-density lipoproteins (HDL). 16 Additionally, AFM may have the potential to study characteristic properties, such as the rigidity, 20 hydrophilicity, 21 and number of lipoprotein particles, 14 which cannot be evaluated in previously mentioned methods. Though EM can be used to measure the size of lipoprotein, it may not reflect the original size as in vivo, as EM requires lipoprotein in dry state.…”

“…Atomic force microscopy (AFM) has been used to obtain topographical images of sample surfaces, such as single-stranded DNA, 12 liposomes, 13,14 and living cells. 15 Using AFM, the surfaces of nanosized materials in fluids or air can be scanned by cantilever.…”