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.
Highlights
Protein folding and unfolding are complex processes governed by the underlying energy landscape and may exhibit parallel pathways, multiple intermediates and transition states (TSs)[1,2,3]
Steady-state fluorescence and time-resolved fluorescence spectroscopy data confirmed that the biophysical properties of azurin were unchanged in the heptamer form (Figs S2–S3 and Table S1)
Single-molecule force spectroscopy (SMFS) experimental data on the heptamers of apo-azurin is shown in Fig. 1
Summary
Protein folding and unfolding are complex processes governed by the underlying energy landscape and may exhibit parallel pathways, multiple intermediates and transition states (TSs)[1,2,3]. Single-molecule force spectroscopy (SMFS) and steered molecules dynamics (SMD) have previously been used to study metalloproteins and probe the effect of metal on the mechanical stability of proteins. Induced by copper-binding on the protein unfolding energy landscape could not be directly ascertained The authors interpreted their results using a model wherein the 2-state pathway was assigned to the apo-protein and the 3-state and 4-state pathways were attributed to the holo-azurin[21]. Through a study of the mechanical unfolding of apo- and holo-proteins separately using SMFS measurements and SMD simulations, we directly probe the copper-dependent changes in the mechanical unfolding pathways of azurin. In the 4-state unfolding pathway, a second intermediate with copper-sequestered β-strands is formed, which unravels to produce the third TS leading to complete unfolding of the protein
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