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De novo design of conformationally flexible transmembrane peptides driving membrane fusion.

ABSTRACT: Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of alpha-helix-promoting Leu and beta-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an alpha-helical state to a beta-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu/Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity.

SUBMITTER: Hofmann MW

PROVIDER: S-EPMC522031 | biostudies-other | 2004 Oct

REPOSITORIES: biostudies-other

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Project description:Self-association is a common phenomenon in biology and one that can have positive and negative impacts, from the construction of the architectural cytoskeleton of cells to the formation of fibrils in amyloid diseases. Understanding the nature and mechanisms of self-association is important for modulating these systems and in creating biologically-inspired materials. Here, we present a two-stage de novo peptide design framework that can generate novel self-associating peptide systems. The first stage uses a simulated multimeric template structure as input into the optimization-based Sequence Selection to generate low potential energy sequences. The second stage is a computational validation procedure that calculates Fold Specificity and/or Approximate Association Affinity (K*association) based on metrics that we have devised for multimeric systems. This framework was applied to the design of self-associating tripeptides using the known self-associating tripeptide, Ac-IVD, as a structural template. Six computationally predicted tripeptides (Ac-LVE, Ac-YYD, Ac-LLE, Ac-YLD, Ac-MYD, Ac-VIE) were chosen for experimental validation in order to illustrate the self-association outcomes predicted by the three metrics. Self-association and electron microscopy studies revealed that Ac-LLE formed bead-like microstructures, Ac-LVE and Ac-YYD formed fibrillar aggregates, Ac-VIE and Ac-MYD formed hydrogels, and Ac-YLD crystallized under ambient conditions. An X-ray crystallographic study was carried out on a single crystal of Ac-YLD, which revealed that each molecule adopts a ?-strand conformation that stack together to form parallel ?-sheets. As an additional validation of the approach, the hydrogel-forming sequences of Ac-MYD and Ac-VIE were shuffled. The shuffled sequences were computationally predicted to have lower K*association values and were experimentally verified to not form hydrogels. This illustrates the robustness of the framework in predicting self-associating tripeptides. We expect that this enhanced multimeric de novo peptide design framework will find future application in creating novel self-associating peptides based on unnatural amino acids, and inhibitor peptides of detrimental self-aggregating biological proteins.

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De novo design of conformationally flexible transmembrane peptides driving membrane fusion.

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