Role of thermolysin in catalytic-controlled self-assembly of fmoc-dipeptides

In recent years, short peptide self-assembled materials, prepared under the control of the thermolysin catalyst, have been investigated extensively and shown to acquire various morphologies and functions as building blocks for a wide range of biomaterials and device applications. However, the role played by thermolysin in this enzymatically triggered peptide self-assembly is still ambiguous. Herein, we designed a series of Fmoc-dipeptide amphiphiles to explore the catalytic role of thermolysin. The results from our experiments and computational simulations showed that hydrophobicity and amino acid sequences of substrates have a significant correlation with thermolysin actions, including the binding capacity and catalytic efficiency. Specifically, thermolysin favors a specific substrate pattern with a hydrophilic amino acid in the first residue and hydrophobic amino acid in the second residue. Moreover, thermolysin catalyzed reactions are bidirectional and could move toward hydrolysis or condensation based on the design of its diverse substrates (peptides). However, the specificity of the enzyme action lies in the major site of its cleavage, which is the terminal hydrophobic or bulky amino side chains. We designed a two-step reaction, taking advantage of the bidirectional catalytic actions of thermolysin, to modify the sequence of Nα-fluorenylmethoxycarbonyl (Fmoc)-dipeptide from Fmoc-YL-COOH to Fmoc-YY-NH2 and treated with thermolysin, which resulted in the enzyme-catalyzed gel-sol-gel transition. This work has an instructive significance in the regulation of peptide sequences, secondary amino acid structures, morphology, and the mechanical property of self-assembled hydrogels with precise design and control at the molecular level via thermolysin catalysis.