Crowding-Induced Mixing Behavior of Lipid Bilayers: Examination of Mixing Energy, Phase, Packing Geometry, and Reversibility

Abstract

In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu(2+), chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domains of bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10(-22) to 1.5 × 10(-20) J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. The mixing process was reversible, with Lo domains reappearing after addition of EDTA for NLP removal.

Figure 1

(A) Chemical structures for phospholipids and IDA-lipids. (B) Schematic of nanolipoprotein particle (NLP) synthesis and its structure.

Figure 2

(A) A representative GUV in the presence of NLP1 exhibiting targeted binding to the L_o domain. GUVs in this sample were synthesized with a lipid composition consisting of 50% DOPC, 30% DPPC, 2% DPIDA, and 18% cholesterol. (B) A representative GUV before and after NLP1 binding exhibiting crowding induced mixing. GUVs in this sample were synthesized with a lipid composition consisting of 50% DOPC, 22% DPPC, 10% DPIDA, and 18% cholesterol. Scale bars are 20 µm.

Figure 3

The percentage of GUVs with visible domains observed in the absence of protein as well as the presence of GFP, NLP1, or NLP3, for various levels of DPIDA and cholesterol content. DPIDA content is represented as relative doping concentration with DPPC. The DOPC:(DPPC+DPIDA) ratio was held constant at 3:2. Vertical error bars represent the standard deviation of triplicate measurements.

Figure 4

The regression curves used to determine ΔF_mix from Equation 8 for various concentrations of cholesterol. The DOPC:(DPPC+DPIDA) ratio was held constant at 3:2. Vertical error bars represent the standard deviation of triplicate measurments.

Figure 5

Representative 50% (DOPC+DOIDA), 32% DPPC, 18% cholesterol GUVs. (A) GUV with 5% DOIDA/(DOPC+DOIDA) either containing 0.1% Texas Red-DHPE displaying a red L_d domain or (B) after addition of OG-DHPE NLP1 displaying a green domain resulting from binding to an L_d region. (C) GUV with 40% DOIDA/(DOPC+DOIDA) either containing 0.1% Texas Red-DHPE displaying a red L_d domain or (D) after addition of OG-DHPE NLP1 displaying uniform green fluorescence resulting from mixing. Scale bars are 20 µm.

Figure 6

Targeting binding of OG-DHPE-containing NLP1 to a lipid multibilayer (MBL) on mica. The MBL was made using a lipid composition consisting of 50% DOPC, 30% DPPC, 2% DPIDA, 18% cholesterol. The scale bar represents a length of 50 µm.

Figure 7

(A) Time lapse of NLP1 induced lipid mixing in multibilayer (MBL) on mica. (B) The removal of bound NLP1 using 2 mM EDTA and reappearance of L_o domains over time. The lipid composition used was 50% DOPC, 20% DPPC, 12% DPIDA, and 18% cholesterol. The scale bar represents a length of 50µm.