Browsing by Author "Drummond, CJ"

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    Behaviour of single transmembrane peptides during in meso crystallization from the contrast-matched lipidic cubic phase of monoolein
    (Australian Institute of Nuclear Science and Engineering (AINSE), 2018-11-19) van't Hag, L; de Campo, L; Tran, N; Sokolova, AV; Trenker, R; Call, M; Garvey, CJ; Leung., A; Darwish, TA; Krause-Heuer, AM; Knott, RB; Meikle, T; Gras, S; Drummond, CJ; Mezzenga, R; Conn, C
    In meso membrane protein crystallization within a lipidic mesophase has revolutionized the structural biology of integral membrane proteins (IMPs). High-resolution structures of these proteins are crucial to understanding fundamental cellular processes at a molecular level, and can lead to new and improved treatments for a wide range of diseases via rational drug design. However, overall success rates of the promising in meso crystallization technique remain low because of a fundamental lack of understanding about factors that promote crystal growth. In particular, to date, two decades from invention of the method, the protein-eye-view of the in meso crystallization mechanism had not been solved. We have investigated this for the first time using small-angle neutron scattering (SANS). Contrast-matching between the scattering of the lipid membrane formed by MO and the aqueous solution was used to isolate and track the scattering of single-transmembrane peptides during the growth of protein crystals in meso. No peptide enrichment was observed at the flat points of the diamond cubic QIID phase of MO in contrast to suggestions in several modeling studies. During in meso crystallization of the DAP12 peptide a decrease in form factor and a transient fluid lamellar Lα phase could be observed providing direct evidence for the proposed crystallization mechanism. Synthesis of fully deuterated MO was required for this purpose and scattering of this new material in various solvents and under a range of conditions will be described, specifically regarding the effect of the relative scattering length densities (SLD) of the headgroup, acyl chain and solvent, which can advance the use of neutron scattering with other self-assembly materials. © The Authors.
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    Determining the location of encapsulated peptides, proteins, and other biomolecules in contrast-matched lipid bicontinuous cubic phases using SANS
    (International Conference on Neutron Scattering, 2017-07-12) van't Hag, L; de Campo, L; Gras, S; Drummond, CJ; Conn, C
    To evolve biological and biomedical applications of hybrid biomolecule?lipid materials, including in meso protein crystallization and drug delivery, an understanding of the location of the biomolecules within the bicontinuous cubic phases is crucial. Theoretical modeling has indicated that proteins and additive lipids might phase separate locally and adopt a preferred location in the cubic phase, but this has never been experimentally confirmed. We have shown that perfectly contrast-matched cubic phases in D2O can be studied using SANS by mixing fully deuterated and hydrogenated lipid. The model transmembrane peptide WALP21 showed no preferential location in the membrane of the diamond and gyroid cubic phases of phytanoyl monoethanolamide [L. van ‘t Hag et al, J. Phys. Chem. Lett. 2016, 7(14), 2862-2866]. In addition, this result opens up the possibility of studying the conformation (and hence function) of amphiphilic proteins and peptides within a lipid bilayer environment. The effect of deuteration on the cubic phase forming lipid was also investigated to advance the use of neutron scattering techniques to study soft matter systems. Additionally, we have performed extensive characterizations of the cubic phase nanostructure subsequent to protein and peptide incorporation using synchrotron SAXS, as well as the protein and peptide secondary structures using synchrotron circular dichroism spectroscopy [L. van ‘t Hag et al, Langmuir, 2016, 32(27), 6882-6894].
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    Transfer of lipid between triglyceride dispersions and lyotropic liquid crystal nanostructured particles using time-resolved SAXS
    (Royal Society of Chemistry., 2012-04-16) Tilley, AJ; Dong, YD; Chong, JYT; Hanley, TL; Kirby, N; Drummond, CJ; Boyd, BJ
    Lipid-based lyotropic liquid crystalline nanostructured phases, such as the reverse bicontinuous cubic phase (V-2) and the reverse hexagonal phase (H-2), can be dispersed into submicron sized particles with often preserved internal nanostructure, termed cubosomes and hexosomes respectively. The internal nanostructure of the particles imparts desirable characteristics such as the ability to provide sustained release of active ingredients but is susceptible to change upon the incorporation of other lipids used as an active ingredient delivery system. These are complex systems in which the direction of lipid transfer, role of the stabiliser and formation of different phase structures, such as the frustrated H-2 phase are all likely to play a role but are not yet completely understood. Consequently, we have investigated the interparticle transfer of lipids between cubosomes/hexosomes and other dispersed lipids, namely triglycerides and vitamin E acetate emulsions. Time-resolved synchrotron small angle X-ray scattering was used to follow changes to nanostructure, attributed to the transfer of lipids into or out of the cubosomes. It was found that transfer of lipid occurred due to compositional ripening via a micelle-mediated mechanism, evident from the rate of lipid transfer increasing with Pluronic concentration. The rate of transfer of alkanes between liquid crystalline particles and emulsions has been shown previously to depend inversely on chain length. However in the triglyceride systems in the current study, the rate of lipid transfer (trilaurin > tricaprylin > supercooled trimyristin >> crystallised trimyristin and tristearin) did not decrease with increase in the chain length of triglyceride as might be anticipated. The equilibrium phase diagrams of triglyceride + phytantriol in excess water were determined. These diagrams showed that the transfer of phytantriol away from cubosomes to the emulsions, which increases the local triglyceride to phytantriol concentration in the cubosomes, would require formation of H-2 phase more readily for tricaprylin than for trilaurin. Therefore, the lack of correlation of transfer rate with chain length of the triglyceride was attributed to the system seeking to avoid the unfavourable formation of the H-2 phase, competing with the driving force from the entropy of mixing, and was therefore attributed as the reason for the greater rate of transfer of trilaurin compared to the less lipophilic tricaprylin. © 2012 Royal Society of Chemistry