Reactivity at the membrane interface

Modulation of internal environment and maintenance of cellular structure and stability are basic requirements to ensure cell survival. These cellular functions are provided by the cell outer membrane, a phospholipid bilayer characterised by the fluid mosaic model. Chemical reactivity at the membrane interface has previously been identified between phospholipids and membrane binding species. Observed reactivity, termed intrinsic lipidation, involves non-enzymatic acyl transfer from phospholipids to a nucleophilic membrane bound molecule. Reactivity has been characterised for membrane active peptides and proteins, and been found influential to the structure and function of both the newly modified species, and the bulk membrane. Research presented within this thesis probes fundamental features of observed intrinsic lipidation reactivity at the membrane interface. This work has expanded upon previous intrinsic lipidation research, facilitated by the development of informative and robust analytical techniques for the study of reactivity. Optimised TLC has allowed improved routine high throughput reactivity screening, compared to alternative fluorescence and solution state NMR techniques. Informative analysis and mechanistic understanding of intrinsic lipidation has been achieved through LCMS and solid state NMR analysis. Synthetic protocols for preparation of isotopically labelled 15N small molecules, and 13C phospholipids, has facilitated solid state NMR in particular. Biological relevance of peptide intrinsic lipidation has also been probed to determine the role of reactivity in natural function, and disease induction. Biophysical techniques such as CD and tryptophan fluorescence revealed that solution phase intrinsically lipidated melittin adopts an α-helical structure with central proline kink, in contrast to the random coil of unmodified melittin. Furthermore, at μM concentrations, palmitoylated species were shown to undergo spontaneous micelle formation. Disease related behaviour linked to peptide intrinsic lipidation includes moderate antimicrobial activity, and possible induction of amyloid nucleation. Additionally, this study has identified novel intrinsic lipidation of small molecules in vitro utilising chromatographic and ionisation conditions optimised with synthetically prepared standards. Observed for multiple cationic amphiphilic small molecules, intrinsic lipidation was promoted by primary amines in a hydrophilic environment, due to increased proximity between reactive moieties. Small molecule intrinsic lipidation products were shown to exhibit biological relevance, including spontaneous micelle formation, membrane disruption, and phospholipidosis induction. Pharmaceutical propranolol displayed notable intrinsic lipidation in vitro, and in Hep G2 cell culture. Initial transesterification from membrane phospholipids produced O-acylated propranolol, followed by secondary N-acylated propranolol formation by intramolecular O to N migration. Study of propranolol reactivity has revealed preferential eukaryotic transfer from the sn-1 phospholipid backbone position, and reaction kinetics influenced by temperature, pH, and membrane composition