Abstract
Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with β-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where β2Β-β8 and β7-β8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length leading to the first intermediate is 6.7 nm suggesting a sequestration of ~1 nm polypeptide chain length by the copper. SMD simulations reveal atomistic details of the copper sequestration and predict a combined β4-β7 pair and copper coordination sphere rupture to create the third TS in the 4-state pathway. Our systematic studies provide detailed mechanistic insights on modulation of protein mechanical properties by metal-cofactors.