Abstract
Lipid-protein interactions can function as “co-factors” that affect the properties / function of transmembrane proteins. Herein, the interaction between anionic dimyristoylphosphatidylglycerol (DMPG) and zwitterionic dimyristoylphosphatidylcholine (DMPC) with the amphiphatic membrane scaffold protein (MSP), were studied. Two 25 kDa MSP wrap around the circumference of discoidal bilayer in a belt-like manner to form a nanodisc [1,2]. The membrane-like structure of nanodiscs has been used for reconstitution of membrane proteins in a native-like environment. Differential scanning calorimetry was employed to characterize lipid-protein interactions in these particles by evaluating changes in MSP denaturation temperature and lipid gel-liquid phase transition as a function of nanodisc lipid composition and ionic strength. Small-angle X-ray scattering and size-exclusion chromatography were used to determine the overall structure of the nanodisc. We suggest the nanodisc lipid is divided into a lipid rim that interacts with the internal face of the MSP helical segments, while the centrally located nanodisc lipids maintain a more bulk-like lipid behavior. This finding is important for reconstitution of membrane proteins since the presence of a ‘lipid rim’ serves to prevent contact between the membrane protein and the MSP. Furthermore, the presence of two distinct lipid environments reduces the available area for reconstituted membrane proteins in the nanodisc. We also show that the negatively charged DMPG has a higher preference for the rim due to its negatively charged headgroup. Finally, we conclude that DMPG stabilizes the nanodisc in a twofold manner: i) DMPG ’freezes’ the MSP conformation preventing flexibility / dissociation that may lead to aggregation. ii) DMPG also contributes to prevention of aggregation due to electrostatic repulsion between the negatively charged lipids on neighboring nanodiscs.[1] T.H. Bayburt et al.: Nano Letters 2 (2002) 853.[2] N. Skar-Gislinge and J.B. Simonsen et al.: JACS 132 (2010) 13713.
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