Role of Zero-Order Loop in Protein Unfolding Case Study with Apoazurin

Abstract

Pseudomonas aeruginosa apoazurin (apo, without the copper cofactor) is a two-state folder that has a single disulfide bond between residues 3 and 26. This bond covalently connects the N-termini of beta-strands 1 and 3; thereby it creates a zero-order loop. The loop restricts the conformational space for the apoazurin polypeptide. In order to understand the role played by the zero-order loop, we used molecular dynamics (MD) simulations to compare two variants of apoazurin; one variant called “loop” which contained the disulfide and another called “open” in which the disulfide bond between residues 3 and 26 was removed. MD simulations were performed to probe the unfolding pathway and stability of the two apoazurin variants at different urea concentrations and temperatures. Our results show that the folded structure apoazurin is somewhat more stable due to the presence of the disulfide bond. However, the disulfide bond plays a prominent role in the apoazurin unfolding mechanism: we find that it changes both the folding-transition state and the unfolded-state ensemble of conformations.