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Tailoring Interleaflet Lipid Transfer with a DNA-based Synthetic Enzyme.

ABSTRACT: Lipid membranes, enveloping all living systems, are of crucial importance, and control over their structure and composition is a highly desirable functionality of artificial structures. However, the rational design of protein-inspired systems is still challenging. Here we have developed a highly functional nucleic acid construct that self-assembles and inserts into membranes, enabling lipid transfer between inner and outer leaflets. By designing the structure to account for interactions between the DNA, its hydrophobic modifications, and the lipids, we successfully exerted control over the rate of interleaflet lipid transfer induced by our DNA-based enzyme. Furthermore, we can regulate the level of lipid transfer by altering the concentration of divalent ions, similar to stimuli-responsive lipid-flipping proteins.

SUBMITTER: Sobota D

PROVIDER: S-EPMC7291347 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Tailoring Interleaflet Lipid Transfer with a DNA-based Synthetic Enzyme.

Sobota Diana D Joshi Himanshu H Ohmann Alexander A Aksimentiev Aleksei A Keyser Ulrich F UF

Nano letters 20200512 6

Lipid membranes, enveloping all living systems, are of crucial importance, and control over their structure and composition is a highly desirable functionality of artificial structures. However, the rational design of protein-inspired systems is still challenging. Here we have developed a highly functional nucleic acid construct that self-assembles and inserts into membranes, enabling lipid transfer between inner and outer leaflets. By designing the structure to account for interactions between ...[more]

PMID: 32374167

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Project description:Using Förster resonance energy transfer, raft/liquid-ordered-domain formation was assessed in asymmetric vesicles containing outer leaflets composed of high-Tm (melting temperature) saturated phosphatidylcholines (diC18:0PC, diC16:0PC, diC15:0PC, or diC14:0PC), low-Tm unsaturated dioleoylphosphatidylcholine (DOPC) and cholesterol, and inner leaflets composed of lipids that by themselves would not form ordered domains (DOPC and cholesterol). Ordered-domain formation in the outer leaflet was compared to that in symmetric vesicles with the same lipid composition as the asymmetric vesicle outer leaflets. The difference between ordered-domain thermal stability in asymmetric and symmetric vesicles was highly dependent on high-Tm PC acyl-chain length. At one extreme, in diC14:0PC-containing asymmetric vesicles, the outer leaflet did not segregate to form ordered domains over the entire experimental temperature range even though ordered domains formed in the symmetric vesicles, indicating the inner leaflet dominated outer-leaflet physical behavior in the asymmetric vesicles. At the other extreme, in diC18:0PC-containing asymmetric vesicles, ordered domains formed over the entire temperature range at which they were present in symmetric vesicles, indicating the inner leaflet did not dominate outer-leaflet physical behavior. DiC15:0PC- and diC16:0PC-containing vesicles exhibited intermediate behaviors. A different set of vesicles was prepared with high-Tm lipid sphingomyelin (SM) in place of saturated phosphatidylcholine, and the % SM was varied. The thermal stability of outer-leaflet ordered domains in asymmetric vesicles was found to decrease more than in symmetric vesicles as SM levels decreased, indicating that the inner leaflet increasingly dominated outer leaflet physical state as SM levels decreased. Overall, inhibition of outer-leaflet ordered-domain formation in asymmetric vesicles by inner-leaflet lipids decreased as the ability of outer-leaflet lipids to form an ordered state by themselves increased, i.e., when outer-leaflet high-Tm lipid content or acyl-chain length increased. This has implications for how ordered-domain formation may be controlled in vivo.

Project description:The immediate immune response to infection by Gram-negative bacteria depends on the structure of a lipopolysaccharide (LPS, also known as endotoxin), a complex glycolipid constituting the outer leaflet of the bacterial outer membrane. Recognition of picomolar quantities of pathogenic LPS by the germ-line encoded Toll-like Receptor 4 (TLR4) complex triggers the intracellular pro-inflammatory signaling cascade leading to the expression of cytokines, chemokines, prostaglandins and reactive oxygen species which manifest an acute inflammatory response to infection. The "endotoxic principle" of LPS resides in its amphiphilic membrane-bound fragment glycophospholipid lipid A which directly binds to the TLR4·MD-2 receptor complex. The lipid A content of LPS comprises a complex mixture of structural homologs varying in the acylation pattern, the length of the (R)-3-hydroxyacyl- and (R)-3-acyloxyacyl long-chain residues and in the phosphorylation status of the β(1→6)-linked diglucosamine backbone. The structural heterogeneity of the lipid A isolates obtained from bacterial cultures as well as possible contamination with other pro-inflammatory bacterial components makes it difficult to obtain unambiguous immunobiological data correlating specific structural features of lipid A with its endotoxic activity. Advanced understanding of the therapeutic significance of the TLR4-mediated modulation of the innate immune signaling and the central role of lipid A in the recognition of LPS by the innate immune system has led to a demand for well-defined materials for biological studies. Since effective synthetic chemistry is a prerequisite for the availability of homogeneous structurally distinct lipid A, the development of divergent and reproducible approaches for the synthesis of various types of lipid A has become a subject of considerable importance. This review focuses on recent advances in synthetic methodologies toward LPS substructures comprising lipid A and describes the synthesis and immunobiological properties of representative lipid A variants corresponding to different bacterial species. The main criteria for the choice of orthogonal protecting groups for hydroxyl and amino functions of synthetically assembled β(1→6)-linked diglucosamine backbone of lipid A which allows for a stepwise introduction of multiple functional groups into the molecule are discussed. Thorough consideration is also given to the synthesis of 1,1'-glycosyl phosphodiesters comprising partial structures of 4-amino-4-deoxy-β-L-arabinose modified Burkholderia lipid A and galactosamine-modified Francisella lipid A. Particular emphasis is put on the stereoselective construction of binary glycosyl phosphodiester fragments connecting the anomeric centers of two aminosugars as well as on the advanced P(III)-phosphorus chemistry behind the assembly of zwitterionic double glycosyl phosphodiesters.

Dataset Information

Tailoring Interleaflet Lipid Transfer with a DNA-based Synthetic Enzyme.

Publications

Tailoring Interleaflet Lipid Transfer with a DNA-based Synthetic Enzyme.

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