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 approximately 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 structure.
Highlights
Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs)
Using atomic force microscopy (AFM) imaging of single NLPs, we have shown that the self-assembly of E422k/DMPC NLPs results in particle size distributions that display discrete diameters centered at 14.7, 18.8, 23.3, and 28.7 nm (Table 1)
transmission electron microscopy (TEM) and ion mobility spectroscopy (IMS) verified these AFM results, eliminating the possibility that these quantized peaks observed by AFM are due to either a tip convolution effect or tip-sample interactions (Fig. 4)
Summary
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. The novel observations in this earlier work, no studies have been done subsequently to examine size heterogeneity for NLPs assembled from engineered lipoprotein constructs for use in biotechnology applications. This concept has not previously been systematically examined using higher resolution particle-sizing techniques
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