Membrane-Catalyzed Aggregation of Islet Amyloid Polypeptide Is Dominated by Secondary Nucleation.

Type II diabetes is characterized by the loss of pancreatic ß-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, that is, the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent ß-cell death in type II diabetes.

Synthesis and Evaluation of a Library of Alternating Amphipathic Copolymers to Solubilize and Study Membrane Proteins.

Amphipathic copolymers such as poly(styrene-maleic acid) (SMA) are promising tools for the facile extraction of membrane proteins (MPs) into native nanodiscs. Here, we designed and synthesized a library of well-defined alternating copolymers of SMA analogues in order to elucidate polymer properties that are important for MP solubilization and stability. MP extraction efficiency was determined using KcsA from E. coli membranes, and general solubilization efficiency was investigated via turbidimetry experiments on membranes of E. coli, yeast mitochondria, and synthetic lipids. Remarkably, halogenation of SMA copolymers dramatically improved solubilization efficiency in all systems, while substituents on the copolymer backbone improved resistance to Ca2+. Relevant polymer properties were found to include hydrophobic balance, size and positioning of substituents, rigidity, and electronic effects. The library thus contributes to the rational design of copolymers for the study of MPs.

Iterative RAFT-Mediated Copolymerization of Styrene and Maleic Anhydride toward Sequence- and Length-Controlled Copolymers and Their Applications for Solubilizing Lipid Membranes.

The use of poly(styrene-co-maleic acid) (SMA) for the solubilization of lipid membranes and membrane proteins is becoming more widespread, and with this, the need increases to better understand the chemical properties of the copolymer and how these translate into membrane solubilization properties. SMA comes in many different flavors that include the ratio of styrene to maleic acid, comonomer sequence distribution, average chain length, dispersity, and potential chemical modifications. In this work, the synthesis and membrane active properties are described for 2:1 (periodic) SMA copolymers with Mw varying from ∼1.4 to 6 kDa. The copolymers were obtained via an iterative RAFT-mediated radical polymerization. Characterization of these polymers showed that they represent a well-defined series in terms of chain length and overall composition (FMAnh ∼ 0.33), but that there is heterogeneity in comonomer sequence distribution (FMSS ∼ 0.50) and some dispersity in chain length (1.1 < D < 1.6), particularly for the larger copolymers. Investigation of the interaction of these polymers with phosphatidylcholine lipid self-assemblies showed that all copolymers inserted equally effectively into lipid monolayers, independent of the copolymer length. Nonetheless, smaller polymers were more effective at solubilizing lipid bilayers into nanodiscs, possibly because longer polymers are more prone to become intertwined with each other, thereby hampering their solubilization efficiency. Nanodisc sizes were independent of the copolymer length. However, nanodiscs formed with larger copolymers were found to undergo slower lipid exchange, indicating a higher stability. The results highlight the usefulness of having well-defined copolymers for systematic studies.

Host defence peptide plectasin targets bacterial cell wall precursor lipid II by a calcium-sensitive supramolecular mechanism.

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates.

Fibril elongation by human islet amyloid polypeptide is the main event linking aggregation to membrane damage.

The aggregation of human islet amyloid polypeptide (hIAPP) is linked to the death of pancreatic ß-cells in type II diabetes. The process of fibril formation by hIAPP is thought to cause membrane damage, but the precise mechanisms are still unclear. Previously, we showed that the aggregation of hIAPP in the presence of membranes containing anionic lipids is dominated by secondary nucleation events, which occur at the interface between existing fibrils and the membrane surface. Here, we used vesicles with different lipid composition to explore the connection between hIAPP aggregation and vesicle leakage. We found that different anionic lipids promote hIAPP aggregation to the same extent, whereas remarkably stochastic behaviour is observed on purely zwitterionic membranes. Vesicle leakage induced by hIAPP consists of two distinct phases for any of the used membrane compositions: (i) an initial phase in which hIAPP binding causes a certain level of leakage that is strongly dependent on osmotic conditions, membrane composition and the used dye, and (ii) a main leakage event that we attribute to elongation of hIAPP fibrils, based on seeded experiments. Altogether, our results shed more light on the relationship between hIAPP fibril formation and membrane damage, and strongly suggest that oligomeric intermediates do not considerably contribute to vesicle leakage.