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De novo self-assembling collagen heterotrimers using explicit positive and negative design.

ABSTRACT: We sought to computationally design model collagen peptides that specifically associate as heterotrimers. Computational design has been successfully applied to the creation of new protein folds and functions. Despite the high abundance of collagen and its key role in numerous biological processes, fibrous proteins have received little attention as computational design targets. Collagens are composed of three polypeptide chains that wind into triple helices. We developed a discrete computational model to design heterotrimer-forming collagen-like peptides. Stability and specificity of oligomerization were concurrently targeted using a combined positive and negative design approach. The sequences of three 30-residue peptides, A, B, and C, were optimized to favor charge-pair interactions in an ABC heterotrimer, while disfavoring the 26 competing oligomers (i.e., AAA, ABB, BCA). Peptides were synthesized and characterized for thermal stability and triple-helical structure by circular dichroism and NMR. A unique A:B:C-type species was not achieved. Negative design was partially successful, with only A + B and B + C competing mixtures formed. Analysis of computed versus experimental stabilities helps to clarify the role of electrostatics and secondary-structure propensities determining collagen stability and to provide important insight into how subsequent designs can be improved.

SUBMITTER: Xu F

PROVIDER: S-EPMC2853261 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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De novo self-assembling collagen heterotrimers using explicit positive and negative design.

Xu Fei F Zhang Lei L Koder Ronald L RL Nanda Vikas V

Biochemistry 20100301 11

We sought to computationally design model collagen peptides that specifically associate as heterotrimers. Computational design has been successfully applied to the creation of new protein folds and functions. Despite the high abundance of collagen and its key role in numerous biological processes, fibrous proteins have received little attention as computational design targets. Collagens are composed of three polypeptide chains that wind into triple helices. We developed a discrete computational ...[more]

PMID: 20170197

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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.

Dataset Information

De novo self-assembling collagen heterotrimers using explicit positive and negative design.

Publications

De novo self-assembling collagen heterotrimers using explicit positive and negative design.

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