Lipid Concentration and Molar Ratio Boundaries for the Use of Isotropic Bicelles

Abstract

Bicelles are model membranes generally made of long-chain dimyristoylphosphatidylcholine (DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensively used in the study of membrane interactions and structure determination of membrane-associated peptides, since their composition and morphology mimic the widespread PC-rich natural eukaryotic membranes. At low DMPC/DHPC (q) molar ratios, fast-tumbling bicelles are formed in which the DMPC bilayer is stabilized by DHPC molecules in the high-curvature rim region. Experimental constraints imposed by techniques such as circular dichroism, dynamic light scattering, or microscopy may require the use of bicelles at high dilutions. Studies have shown that such conditions induce the formation of small aggregates and alter the lipid-to-detergent ratio of the bicelle assemblies. The objectives of this work were to determine the exact composition of those DMPC/DHPC isotropic bicelles and study the lipid miscibility. This was done using 31P nuclear magnetic resonance (NMR) and exploring a wide range of lipid concentrations (2–400 mM) and q ratios (0.15–2). Our data demonstrate how dilution modifies the actual DMPC/DHPC molar ratio in the bicelles. Care must be taken for samples with a total lipid concentration ≤250 mM and especially at q ∼ 1.5–2, since moderate dilutions could lead to the formation of large and slow-tumbling lipid structures that could hinder the use of solution NMR methods, circular dichroism or dynamic light scattering studies. Our results, supported by infrared spectroscopy and molecular dynamics simulations, also show that phospholipids in bicelles are largely segregated only when q > 1. Boundaries are presented within which control of the bicelles’ q ratio is possible. This work, thus, intends to guide the choice of q ratio and total phospholipid concentration when using isotropic bicelles.

Characterization of Small Isotropic Bicelles with Various Compositions

“…(5)-(9) resulted in the following parameters: diffusion coefficient at infinite dilution, D o =111±1x10 -12 m 2 s -1 , bicelle radius, R=3.1±0.1 nm, ‫]ݐ݁ܦ[‬ =8.3±0.3 mM at q*=0.53 and 40°C (Table S1). According to DLS measurements, q=0.5 bicelles have radii in the range 2.9-3.3 nm, depending on concentration 45 and phosphorous NMR yields ‫]ݐ݁ܦ[‬ in the range 7-8 mM for this particular q ratio 22,46 . However, it is noteworthy that the results of other NMR diffusion studies are very controversy.…”

“…MD investigation reveals that pre-assembled q=0.25 bicelles are not stable during the simulations 46 , experimental works claim that parameters of small bicelles differ a lot from the parameters of magnetically oriented bicelles: they do not demonstrate any lipid phase transition, or phase transition is not expressed and have shifted critical temperature 45,46 ; size of isotropic bicelles deviates from predicted by the models used in some works. Therefore, the authors conclude that at low q DMPC/DHPC mixtures form mixed micelles rather than bicelles.…”

“…A recent work reports that DMPC/DHPC bicelles grow in size upon dilution 46 . This effect was attributed to the presence of certain amount of free DHPC in solution, which does not depend on the concentration of bicelles and increases the effective q* ratio.…”

Magnetically Oriented Bicelles with Monoalkylphosphocholines: Versatile Membrane Mimetics for Nuclear Magnetic Resonance Applications

“…Indeed, the presence of a significant amount of free surfactant can be a drawback in particular when samples need to be diluted 42 . By studying the effect of dilution on 31 P NMR spectra, the concentration of free detergent (or critical bicellar concentration, CBC) can be determined 41,42 . Examples of such spectra are shown in the supporting information ( Figure S6), and CBC values are listed in Table 1, calculated as described by Beaugrand et al 42 .…”

“…For a given molar ratio q of 1 and detergent 12PC, the CBC for D12PC, D14PC and D16PC are 0.75, 0.78 and 0.82 mM, respectively. By increasing the length of the phospholipid, the CBC slightly increases, indicating a decrease in miscibility when the chain-length mismatch increases 42 . For a given detergent 12PC and phospholipid D14PC, the CBC slightly decreases when q increases from 0.5 to 1, an effect also observed for classical bicelles and attributed to a higher segregation between D14PC and D6PC as q increases 42 .…”