The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

Abstract

Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.

Ab initio electronic structure calculations of entire blue copper azurins

“…61 This force-field includes both bonded and non-bonded terms between the Cu atom and its 5 ligands and has been widely used to model the blue-copper Azurin protein. 24,42,[62][63][64] In particular, recent experiments 63 have shown how early stages of mechanical unfolding of this protein are well described by this force-field. The X-ray crystallographic structure of Azurin was obtained from the protein data bank 65 with the PDB code 4AZU.…”

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

“…The field has advanced, thanks to the development of instrumentation and the methods to analyse and interpret the data [245][246][247]. SMFS is now embedded within the scientific community and is being exploited to probe a diverse range of biological systems, including proteins, DNA, RNA and their complexes [67,81,85,157,[248][249][250][251][252]. While early experiments provided the first measurements of protein mechanical stability, further experimental and theoretical studies have pin-pointed the molecular origin of this resilience.…”

CHAPTER 1.3. Real-time Detection of Thiol Chemistry in Single Proteins

“…To prove the existence of the S-sulfenylated intermediate, Beedle et al performed experiments at ph 12.8 in the presence of 30 mM dimedone, which reacts irreversibly with sulfenic acid, but not any other oxidation state of the cysteine side-chain. 57 The bulky dimedone side-group blocked protein folding of I91 (24C/55C) in single-molecule experiments and its presence was also confirmed using mass spectrometry. There are several important differences between non-enzymatic oxidative folding and what is seen with the oxidoreductase enzymes.…”

“…Building on the previously discussed studies of disulfide cleavage by the hydroxyl ion (oh − ), Beedle et al used this nucleophile to explore non-enzymatic reoxidation of protein disulfide bonds. 57 Instead of using oxidized I91 (32C/75C) as the substrate, the engineered I91 (24C/55C) was used in these studies. This disulfide bond cannot be accessed by hydroxyl ions when the protein is in its folded state as all disulfides seem to be formed during the initial denature pulse.…”

“…Similarly to pdI and dsbA, the hydroxyl ion acts as a placeholder for the cleaved disulfide bond by formation of an S-sulfenylated (-Soh) intermediate. 57 when the force on the protein is quenched during the refolding pulse, the free thiol of I91 can attack the sulfenic moiety via an S n 2 reaction, releasing the hydroxyl ion back into solution and re-forming the disulfide bond. however, the -Soh group is short lived and can progress to the higher thiol oxidation states of sulfinic and sulfonic acids.…”