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Cholesterol Organization in Phosphatidylcholine Liposomes: A Surface Plasmon Resonance Study

ABSTRACT: Models for the organization of sterols into regular arrays within phospholipid bilayers have been proposed previously. The existence of such arrays in real systems has been supported by the fact that concentration-dependent sterol properties show discontinuities at the cholesterol mole fractions corresponding to regular lattice arrangements. Experimental results presented here are based on a surface plasmon resonance assay that was used to analyze rates of cyclodextrin-mediated removal of cholesterol from adsorbed liposomes at cholesterol mole fractions up to χC = 0.55. Two kinetic pools of cholesterol were detected; there was a fast pool present at χC > 0.25, and a slow pool, with a removal rate that was dependent on the initial χC but that did not vary as χC decreased during the course of one experiment. The cholesterol activity therefore seems to be affected by sample history as well as local concentration, which could be explained in terms of the formation of superlattices that are stable for relatively long times. We also describe a variation on the traditional lattice models, with phosphatidylcholine (PC) being treated as an arrangement of hexagonal tiles; the cholesterol is then introduced at any vertex point, without increasing the total area occupied by all the lipid molecules. This model is consistent with Langmuir trough measurements of total lipid area and provides a simple explanation for the maximum solubility of cholesterol in the PC bilayer.

SUBMITTER: Melzak K

PROVIDER: S-EPMC5448994 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

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Cholesterol Organization in Phosphatidylcholine Liposomes: A Surface Plasmon Resonance Study

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