(C-91) Temperature Modulates the Assembled Structure of Co-Assembling Synthetic Peptides

Introduction:: Supramolecular biomaterials are highly tunable as they assemble using non-covalent interactions between monomeric species that may break and reform. In addition, supramolecular biomaterials are highly responsive to their environment and may develop unique architectures or properties when parameters such as monomer concentration, temperature, and ionic strength are changed. Peptides that associate non-covalently are a prime monomer for supramolecular biomaterials due to the numerous natural and synthetic amino acids that can be combined, offering a great deal of modification potential. One such peptide-based supramolecular biomaterial is the CATCH (Co-Assembling Tags based on CHarge complementarity) system, a co-assembling system using a pair of positively and negatively charged 11-residue peptides with hydrophobic motifs to generate b-sheet nanofibers that form gels at sufficiently high concentration¹. One of the most studied CATCH peptide pairs is the CATCH(4K+/6E-) pair with a cationic peptide containing 4 lysine residues and an anionic residue containing 6 glutamate residues. The high charge of each peptide prevents self-association in aqueous solution, but CATCH(4K+/6E-) rapidly co-assembles to form b-sheet nanofibers when mixed in equimolar ratios. Prior work has characterized the assembly of CATCH(4K+/6E-) at room temperature, but there is a need to the material response to environmental perturbation to more effectively design supramolecular materials with the desired properties.


Materials and Methods:: CATCH Nanofiber Fabrication: Positively charged CATCH(+) and negatively charged CATCH(-) peptides were dissolved in aqueous buffer, combined in equimolar ratios, and maintained at the specified temperature. Thioflavin T (ThT) Assay: Peptides were mixed with ThT for final peptide concentration of 700 µM and 0.08 mg ThT/mL. Circular Dichroism (CD): Peptides were prepared at 50-400μM in chloride-free sodium phosphate buffer. Transmission Electron Microscopy (TEM): Peptide solutions were prepared at 100-700 µM and adsorbed onto Formvar/carbon grids before negative staining with 2% uranyl acetate.


Results, Conclusions, and Discussions:: Prior studies of CATCH(4K+/6E-) show a transition from unstructured secondary structure at low concentrations to a primarily b-sheet conformation around 300 μM. CD assessment of CATCH(4K+/6E-) secondary structure between 50 and 400 μM showed a correlation between temperature and the concentration of CATCH(4K+/6E-) required to develop b-sheets. At 4°C, random coil structures were observed ≤150 μM and primarily b-sheet ≥300 uM. At 23°C and 37°C, 400 μM was required to achieve b-sheet structure, and at 68°C, all concentrations showed transitional states. Observed secondary structures at room temperature were consistent with prior work¹. These data suggest an increased concentration required for secondary structural transitions at higher temperatures.
TEM analysis of CATCH(4K+/6E-) showed nanofibers at 23°C and 4°C, consistent with previous literature^1–3. At 68°C, few fibers were visible with large aggregates forming.
The quantity of cross-b fibrils were analyzed using ThT. At 68°C, low fibril mass was observed. At 4°C and 23°C, fibrils formed rapidly, consistent with previous reports. ThT measurements were repeated using Q11, a single component analog to CATCH and similar trends were observed, suggesting temperature-dependent trends in fibril formation are not unique to CATCH.

The stability of CATCH(4K+/6E-) fibril assemblies were studied by cycling temperature. Transfer between 23°C and 4°C conditions showed no difference from assembly at the current temperature. ThT signal was sharply attenuated at 68°C with a reduction in total fibril mass formed post-68°C-incubation. CATCH(4K+/6E-) was next either swapped between 68°C and 4°C every hour, or incubated at 68°C for until 4 hours had been spent at 68°C and equilibrated at 4°C afterwards. The final ThT signal in both groups were equivalent, indicating that assembled state convergence depends on the incubation time, not cycle number.

Here, we demonstrate a temperature-mediated increase in the concentration of CATCH(4K+/6E-) required to undergo canonical assembly pathways. Higher temperature necessitates higher concentrations of CATCH(4K+/6E-) to transition to b-sheets, and results in higher CAC. Temperature inversely correlates to fibril mass, at a given concentration of CATCH(4K+/6E-). Additionally, alternative pathways forming non-fibrillar aggregates become possible at higher temperatures.


Acknowledgements (Optional): :

References (Optional): : 1. Seroski, D. T. et al. Co-Assembly Tags Based on Charge Complementarity (CATCH) for Installing Functional Protein Ligands into Supramolecular Biomaterials. Cell. Mol. Bioeng. 9, 335–350 (2016).

2. Shao, Q. et al. Anatomy of a selectively coassembled β-sheet peptide nanofiber. Proc. Natl. Acad. Sci. U. S. A. 117, 4710–4717 (2020).

3. Seroski, D. T. et al. Charge guides pathway selection in β-sheet fibrillizing peptide co-assembly. Commun. Chem. 2020 31 3, 1–11 (2020).