Different Apolipoproteins Impact Nanolipoprotein Particle Formation

Abstract

Spontaneous interaction of purified apolipoproteins and phospholipids results in formation of lipoprotein particles with nanometer-sized dimensions; we refer to these assemblies as nanolipoprotein particles or NLPs. These bilayer constructs can serve as suitable mimetics of biological membranes and are fully soluble in aqueous environments. We made NLPs from dimyristoylphospatidylcholine (DMPC) in combination with each of four different apolipoproteins: apoA-I, Delta-apoA-I fragment, apoE4 fragment, and apolipophorin III (apoLp-III) from the silk moth B. mori. Predominately discoidal in shape, these particles have diameters between 10 and 20 nm, share uniform heights between 4.5 and 5 nm, and can be produced in yields ranging between 40 and 60%. The particular lipoprotein, the lipid to lipoprotein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles and indicate that apoA-I NLP preparations are smaller than the larger apoE422K and apoLp-III NLP preparations.

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

“…NLP formation and purification have been described in detail elsewhere (14). Briefly, dried DMPC was dissolved in 10 mM Tris, pH 7.4, 0.15 M sodium chloride, 0.25 mM EDTA, 0.005% sodium azide (TBS) buffer at a concentration of 20 mg/ml followed by probe sonication to clarity.…”

“…The development of an electrospray interface provides a way to analyze particles suspended in a liquid. We used an electrospray interface (model 3480; TSI, Inc., Shoreview, MN) and a Macroion Mobility Spectrometer (model 3890; TSI, Inc.) to measure the size distribution of NLP particles after they were exchanged via dialysis into a 25 mM ammonium acetate buffer (14,26,27). The methodology used to prepare the samples and measure the NLP size distributions were similar to a method developed by Benner and colleagues (14,26,27) for analyzing human lipoprotein particles and NLPs.…”

“…NLP samples were diluted using TBS, mounted onto carboncoated 400 mesh copper electron microscopy grids, stained with 2% uranyl acetate, and imaged using a Philips CM300 FEG transmission electron microscope operating at an accelerating voltage of 300 keV, as described previously (14).…”

“…This was first demonstrated by Jonas, Drengler, and Patterson (3) using purified apolipoprotein A-I (apoA-I). Since then, the self-assembly of these discoidal nanolipoprotein particles (NLPs) has been demonstrated for a wide range of other apolipoproteins, including apoE, apoC, apoB, and apolipohorin III (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Recently, Sligar and others (1,2,(15)(16)(17) have shown that when membrane proteins are present during NLP self-assembly, these complexes become associated with the particle, aiding in membrane protein solubilization.…”

“…Because SEC, GGE, and SAXS all determine particle size derived from averages of ensembles of particles, none can provide biophysical data capturing subtle differences in particle size from multiple heterogeneous populations. Single-particle sizing techniques, such as TEM, atomic force microscopy (AFM), and ion mobility spectroscopy (IMS), enable quantification of the size of individual NLPs (14), allowing the population size distribution to be examined. Such information has the potential to provide insights into the relationship between NLP size and structural composition and may prove valuable for biotechnology applications of NLPs as model membrane systems for membrane protein solubilization.…”