Smadbeck J, Chan KH, Khoury GA, Xue B, Robinson RC, et al. (2014) De Novo Design and Experimental Characterization of Ultrashort Self-Associating Peptides. PLoS Comput Biol 10(7): e1003718. doi:10.1371/journal.pcbi.1003718
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 β-strand conformation that stack together to form parallel β-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.
CAF acknowledges support from the National Institutes of Health (5R01 GM052032) and the National Science Foundation (CBET-091143). JS acknowledges support from NIH (P50GM071508-06). GAK acknowledges support from a National Science Foundation Graduate Research Fellowship under grant number DGE-1148900. CAEH acknowledges support by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). BX and RCR acknowledge support by the Institute of Molecular and Cell Biology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.