Single‐Molecule Force Spectroscopy Reveals Stability of mitoNEET and its [2Fe2Se] Cluster in Weakly Acidic and Basic Solutions

Abstract

The outer mitochondrial membrane protein mitoNEET (mNT) is a recently identified iron‐sulfur protein containing a unique Fe2S2(His)1(Cys)3 metal cluster with a single Fe−N(His87) coordinating bond. This labile Fe−N bond led to multiple unfolding/rupture pathways of mNT and its cluster by atomic force microscopy‐based single‐molecule force spectroscopy (AFM‐SMFS), one of most common tools for characterizing the molecular mechanics. Although previous ensemble studies showed that this labile Fe−N(His) bond is essential for protein function, they also indicated that the protein and its [2Fe2S] cluster are stable under acidic conditions. Thus, we applied AFM‐SMFS to measure the stability of mNT and its cluster at pH values of 6, 7, and 8. Indeed, all previous multiple unfolding pathways of mNT were still observed. Moreover, single‐molecule measurements revealed that the stabilities of the protein and the [2Fe2S] cluster are consistent at these pH values with only ≈20 pN force differences. Thus, we found that the behavior of the protein is consistent in both weakly acidic and basic solutions despite a labile Fe−N bond.

Mechanical forces and ligand-binding modulatePseudomonas aeruginosaPilY1 mechanosensitive protein

“…Ca 2+ binding to its cognate site in blade IV increases the mechanical stability of the variable intermediate I4 and delays the unfolding of the subsequent domains (Figures 2 and 4, Supplementary Figures 1 and 7). Furthermore, this metal increases the frequency of conformations sparsely visited in EGTA (Figures 2 and 4, and Supplementary Figures 2 to 5), indicating that metal binding impacts the mechanical properties and the unfolding pathway of the protein 30,[45][46][47][48] . The fact that the total unfolding extension of the variable intermediates is always the same confirms that this region of PilY1 folds between unfolding cycles (Figures 2 and 4).…”