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42874-G4,7
Nanostructured Fibrillar Protein Hydrogels Produced via Self-Assembly-Directed Peptide Ligation
Introduction: The research supported by this ACS PRF G-type grant deals with developing novel polymerization techniques for transforming non-covalent assemblies of peptides into mechanically stable hydrogels. Peptides and peptidomimetics that self-assemble into non-covalent networks in water produce hydrogels with attractive features such as stimulus-responsiveness, bioactivity, and predictable fibrillar nanostructures. These properties make them promising candidates for applications including stimulus-sensitive actuation, catalysis, biomaterials, and separations. Because these materials are typically constructed with simple folding motifs (short beta-sheets and alpha-helices), design rules for producing desired nanostructures have been elucidated, and exquisitely controlled fibril morphologies have been recently reported. However, while using short peptides confers control over folding and fibrillar structure, it limits the materials' mechanical properties because the majority of bonds within the fibrils are non-covalent. We are addressing this issue in this PRF-supported project by exploring peptide polymerization via native chemical ligation as a route towards more mechanically stable networks. The templating of polymerization via peptide self-assembly is utilized to favor polymerization over cyclization. We are achieving this through pH control by designing peptides capable of assembling at low pH, where ligation is prevented. Raising pH after peptide assembly then induces polymerization.
Personnel Supported: In year 1, this funding partially supported the PI's salary as well as those of two undergraduate cooperative education students. In year 2, this funding partially supported one undergraduate cooperative education student.
Scientific Progress Report: In the first year of the project, we proved the concept of using ligation polymerization to stiffen peptide gels. In the second and last year of this project we have achieved significant success in understanding the molecular species responsible for this stiffening and the impact of ligation polymerization on the secondary structure and morphology of the self-assembled peptide fibrils. We first developed synthesis and purification methodology for N-terminal cysteine peptide alpha-thioesters. These peptides assemble into beta-sheet fibrillar structures observable by TEM. At low pH, polymerization is prevented, but upon neutralization, polymerization proceeds rapidly via native chemical ligation. Using SDS-PAGE we have optimized the conditions for this reaction and have observed the formation of at least hexamers (9.9kDa) after about 20 minutes. MALDI-TOF mass spectrometry indicated that the polymerized species were mixtures of ligated dimers, trimers, tetramers, cyclic dimers, and cyclic trimers. In 30mM peptide gels, formation of these polymerized products improves storage modulus to about 50kPa (a), which is significantly stiffer than other reported gels produced from similar peptides. In addition, the zero-length cross-link that is produced through native chemical ligation does not disrupt the self-assembled fibril, as assessed by TEM (b), and a predominantly beta-sheet secondary structure is maintained as evidenced by circular dichroism spectroscopy. This method provides a chemoselective route for stiffening peptide hydrogels. It is orthogonal to any additional amino acid sequence intended to confer specific functionality in the gel, so we anticipate that this approach will be useful in future studies in which these matrices are used as multi-functional coatings for interfacing synthetic materials with biological milieus. In addition, this project enabled the collection of preliminary data necessary for a successful NSF-CAREER award submission by the PI, and it provided hands-on research experiences to two undergraduate co-op students.
