A pH Switch for β‐Sheet Protein Folding

Protein design advancements have led to biotechnological strategies based on more stable and more specific structures. Herein we present a 6‐residue sequence (HPATGK) that acts as a stable structure‐nucleating turn at physiological and higher pH but is notably unfavorable for chain direction reversal at low pH. When placed into the turn of a β‐sheet, this leads to a pH switch of folding. Using a standard 3‐stranded β‐sheet model, the WW domain, it was found that the pH switch sequence insertion caused minimal change at pH 8 but a ca. 50 °C drop in the melting temperature (T_m) was observed at pH 2.5: ΔΔG_F ≥11.3 kJ mol⁻¹. Using the strategies demonstrated in this article, the redesign of β‐sheets to contain a global, or local, pH‐dependent conformational switch should be possible.     

Mechanical Deformation Accelerates Protein Ageing

A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week‐long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.