Quantifying size distributions of Nanolipoprotein particles (NLPs) with single particle analysis and molecular dynamic (MD) simulations

Craig . D Blanchette, Richard Law, W. Henry Benner, Joseph B. Pesavento, Jenny A. Cappuccio, Vicki Walsworth, Edward A. Kuhn, Michele Corzett, Brett A. Chromy, Brent W. Segelke, Matthew A. Coleman, Graham Bench, Paul D. Hoeprich, and Todd A. Sulchek

Chemistry, Material, Earth and Life Sciences, Lawrence Livermore National Laboratories, Livermore, CA 94551-9900

Corresponding Author: blanchette2{at}llnl.gov

Self-assembly of purified apolipoproteins and phospholipids results in 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 reagents for biotechnology applications by serving as model membranes to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time and it is believed that size depends on the apolipoprotein chosen for assembly. 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 (AFM), ion mobility spectrometry (IMS) and transmission electron microscopy (TEM) 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 (apoE422k). 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 modeling studies suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs. The initial structural simulations also suggest that particles with an odd number of lipoproteins are consistent with the double belt model where at least one lipoprotein adopts a hairpin structure. In addition, discrete sizes were also observed in NLPs self-assembled from E422k/1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), A-I/DMPC, and commercially obtained NLPs purchased from Nanodisc, Inc. indicating this is likely a general and physically relevant phenomenon.

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