Differences in the mechanical unfolding pathways of apo- and copper-bound azurins

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.

Unfolding pathway and its identifiability in heterogeneous chains of bistable units

“…This concept has been applied to deterministic (linear and exponential) and random distributions of energy jumps. The results of this work can be applied to the better interpretation of the force spectroscopy measurements of biological macromolecules [57][58][59] and to the accurate design of microand nano-systems based on bistable chains with specific properties [60].…”

“…From one side, it allows a better understanding of the unfolding pathways of proteins and other bio-macromolecules, typically measured through force-spectroscopy techniques. In particu-375 lar, it can explain the statistical modifications or variability of the unfolding pathway, which is sometimes depending on several experimental conditions [57][58][59]. On the other side, the identificability concept may be useful to improve the design of heterogeneous micro-and nano-systems based on bi-and multi-380 stability, where folding and unfolding sequences represent the response of the system and should be therefore stable to temperature variations and to other structural or external parameters [60].…”

Toward Cancer Diagnostics of the Tumor Suppressor p53 by Surface Enhanced Raman Spectroscopy

“…As capture molecules for p53, we selected two different kinds of molecules: Az and specific antibodies. The blue copper-containing Az protein (MW 14kDa), whose structure from X-ray investigation is represented in Figure 1 b [ 54 ], is characterized by a high structural stability and well-defined mechanical properties [ 60 , 61 ]. Notably, Az exhibits peculiar redox and optical properties which make it suitable also for bio-nano electronics applications [ 62 , 63 , 64 ].…”

Blue light-induced low mechanical stability of ruthenium-based coordination bonds: an AFM-based single-molecule force spectroscopy study

“…(1) The pulling geometry does not change the transition state, but simply alters the relative position of the transition state along the reaction coordinate; (2) the pulling geometry alters the structure of the transition state, which, in turn, affects both the energy and position of the ratelimiting barrier. 62 To further investigate the molecular mechanism and freeenergy landscape of the Ru II -N dynamic bond, we performed dynamic loading experiments. We changed the loading rate by changing the pulling speed.…”