1. Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles
- Author
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W. Henry Benner, Graham Bench, Paul D. Hoeprich, Jenny A. Cappuccio, Todd Sulchek, Craig D. Blanchette, Brett A. Chromy, Brent W. Segelke, Edward A. Kuhn, and Matthew A. Coleman
- Subjects
Streptavidin ,Nanostructure ,Ion-mobility spectrometry ,Lipoproteins ,Biophysics ,Bacteriorhodopsin ,Microscopy, Atomic Force ,NLP ,Biochemistry ,Atomic force microscopy ,chemistry.chemical_compound ,Particle Size ,Nanodisc ,biology ,Chemistry ,Membrane Proteins ,Cell Biology ,Apolipoprotein ,Nanostructures ,Crystallography ,Membrane protein ,Bacteriorhodopsins ,Biotinylation ,biology.protein ,Spectrophotometry, Ultraviolet ,Particle size ,Nanolipoprotein particle - Abstract
To better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly.
- Published
- 2009