Protein stabilization by tuning the steric restraint at the reverse turn

Area of Science:

• Protein engineering
• Biochemistry
• Structural biology

Background:

• Reverse turns are critical protein motifs for nucleating beta-sheets and driving protein folding.
• Their solvent-exposed nature allows for chemical modifications to enhance protein stability.
• Modulating protein stability is key for re-engineered proteins and de novo protein design.

Purpose of the Study:

• To investigate the use of van der Waals repulsive forces to tune steric restraint at protein reverse turns.
• To develop a novel method for enhancing the stability of beta-sheet structures in proteins.
• To create a versatile toolkit for modulating the stability of beta-sheet containing peptides and proteins.

Main Methods:

• Utilizing N-methylation of the i+1-i+2 amide bond at the reverse turn to induce steric restraint.
• Introducing engineered turn motifs into a three-stranded beta-sheet protein (Pin 1 WW domain).
• Analyzing thermal stability (Tm) and changes in free energy of folding (ΔΔGf) of protein variants.

Main Results:

• N-methylation of reverse turns induced steric restraint, leading to stable, well-folded beta-sheets in aqueous solution.
• Engineered turn motifs were compatible with coded amino acids and bioactive loops.
• Pin 1 WW domain variants exhibited enhanced thermal stability (Tm 62-82 °C) and a cooperative two-state transition.
• Protein stability was modulated by up to ~2.8 kcal mol-1 (ΔΔGf) without perturbing the native fold.

Conclusions:

• A simple loop engineering strategy via two amino acid substitutions provides a toolkit to modulate beta-sheet protein stability.
• Conformational preorganization at the engineered reverse turn leads to enhanced structural stability through intramolecular hydrogen bonds.
• This approach significantly expands the scope of de novo protein and foldamer design in aqueous solutions.