Reversible thermal denaturation of a 60-kDa genetically engineered beta-sheet polypeptide

Area of Science:

• Protein folding dynamics
• Biophysics
• Materials science

Background:

• Understanding protein folding is crucial for deciphering biological processes and developing new biomaterials.
• Beta-sheet structures are fundamental to protein stability and function, yet their formation dynamics are complex.
• De novo designed polypeptides offer controlled systems to investigate folding mechanisms.

Purpose of the Study:

• To characterize the folding properties of a de novo designed polypeptide with a regular repeat sequence.
• To explore beta-sheet formation and fibril assembly using this polypeptide as a model system.
• To gain insights into the folding pathways and kinetics of beta-sheet proteins.

Main Methods:

• Synthesis of a 687-amino acid polypeptide with a specific repeating sequence: (GA)(3)GY(GA)(3)GE(GA)(3)GH(GA)(3)GK.
• Thermal denaturation and refolding experiments.
• Deep ultraviolet Raman spectroscopy to monitor structural changes and intermediates during folding/unfolding.

Main Results:

• The polypeptide forms stable beta-sheet assemblages and fibrillar structures at room temperature.
• Complete and reversible denaturation occurs at 125°C, transitioning to a polyproline II conformation.
• Melting and refolding kinetics are monoexponential, suggesting a lack of detectable hairpin-type nuclei on the millisecond timescale.
• Folding dynamics align with initial collapse preceding secondary structure formation, exhibiting faster-than-expected kinetics.

Conclusions:

• The designed polypeptide serves as an effective model for studying beta-sheet formation and protein folding.
• The folding pathway appears less frustrated, contributing to its rapid folding kinetics.
• The sequence design balances inter-residue interactions, facilitating efficient folding without significant nonnative contacts.