Influence of different polymer belts on lipid properties in nanodiscs characterized by CW EPR spectroscopy

Abstract

With this study we aim at comparing the well-known lipid membrane model system of liposomes and polymer-encapsulated nanodiscs regarding their lipid properties. Using differential scanning calorimetry (DSC) and continuous wave electron paramagnetic resonance (CW EPR) spectroscopy, we characterize the temperature-dependent lipid behavior within 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) liposomes and nanodiscs made from such liposomes by application of various polymers based on styrene-co-maleic acid (SMA), diisobutylene-alt-maleic acid (DIBMA), and styreneco-maleic amide sulfobetaine (SMA-SB), a new SMA-derived copolymer containing sulfobetaine side chains. By incorporating a spin label doxyl moiety into the lipid bilayer in position 16 or 5 we were able to study the micropolarity as well as rotational restrictions onto the lipids in the apolar bilayer center and the chain region adjacent to the carbonyl groups, respectively. Our results suggest that all polymers broaden the main melting transition of DMPC, change the water accessibility within the lipid bilayer, and exhibit additional constraints onto the lipids. Independent of the used polymer, the rotational mobility of both spin-labeled lipids decreased with DIBMA exerting less restraints probably due to its aliphatic side chains. Our findings imply that the choice of the solubilizing polymer has to be considered an important step to form lipid nanodiscs which should be included into research of lipid membranes and membrane proteins in the future.

Mechanisms of Formation, Structure, and Dynamics of Lipoprotein Discs Stabilized by Amphiphilic Copolymers: A Comprehensive Review

“…EPR spectroscopy of spin-label doxyl moieties incorporated into the lipid bilayer in the 5th or 16th position combined with differential scanning calorimetry (DSC) allowed characterizing the temperature-dependent lipid properties in a DMPC model membrane surrounded by SMA, DIBMA, or poly(styrene-co-maleic amide sulfobetaine) (SMA-SB), as compared to liposomes [ 105 ]. The authors found that all three polymers broadened the main melting transition of DMPC, changed the water accessibility within the lipid bilayer, and imposed additional constraints onto the lipids.…”

“…The copolymers exerted steric constraints onto the hydrophobic part of the lipids, while a small loosening effect was observable for the carbonyl-near membrane region [ 106 ]. The authors concluded that the choice of the solubilizing polymer is important in forming lipodiscs [ 105 ].…”

Nanoscale Model System for the Human Myelin Sheath

“…The additional spectral component, featuring increased polarity and hydration in each model system, can be consistently explained with the addition of the amphiphilic polymers, i.e., SMA , and SMA-SB, , that change polarity directly plus disrupt the bilayer structure by providing the basis for increased water penetration into the core of the membrane. ,, On the other hand, increased restrictions of spin-label rotational mobility can either be the result of a polymer-induced formation of a condensed lipid phase , or by lipid–polymer interactions with the surrounding polymer itself. ,, To study this effect, we performed DSC measurements of SMALPs and SMA-SBLPs of both lipid compositions prior to the separation of the liposomes.…”

“…Polymer stock solutions were prepared as previously described. , An appropriate amount of either SMA or SMA-SB was dissolved in saline tris buffer (50 mM tris, 300 mM NaCl, pH 7.4 in Millipore Milli-Q water with a specific resistance of ρ = 18.2 MΩ cm) to yield mass concentrations of 25 mg/mL (SMA) or 15 mg/mL (SMA-SB) at room temperature, followed by heating combined with ultrasonication for 30 min at 70 °C. After cooling down, both polymer stock solutions were stable for 2 weeks at room temperature.…”

“…In this work, we show how nanoscale models for myelin lipid bilayer membranes with the composition of the central nervous system (CNS) or peripheral nervous system (PNS) cytosolic leaflet can be achieved (see the Supporting Information for details on criteria) by solubilization of myelin-like liposomes of the cytosolic CNS and PNS lipid composition with the well-known polymers styrene/maleic acid (SMA 2:1) and diisobutylene/maleic acid (DIBMA) as well as the new polymer styrene/maleimide sulfobetaine (SMA-SB). , The solubilization of these lipid mixtures is remarkably challenging because of the high amount of negatively charged, unsaturated phospholipids, and the exceptionally high cholesterol content. − After preparation, the nanodiscs were investigated regarding their lipid composition using high-performance liquid chromatography (HPLC) and mass spectrometry (MS), their lipid properties with continuous-wave electron paramagnetic resonance (CW EPR), and their interactions with the model protein bMBP, which is known to act as “molecular glue” between two myelin layers in both PNS and CNS . The achievement of lipid nanodiscs with the challenging lipid compositions of PNS and CNS myelin can in general be viewed as paving the way for more complex lipid model membrane systems, on the one hand, and in this specific case allows for future studies on protein–lipid interactions in myelin and factors enabling or even enforcing myelin formation or degradation, even using native-like combinations of myelin proteins in a realistic yet highly controlled lipid environment.…”