Transforming de novo protein α₃D into a mechanically stable protein by zinc binding

Abstract

α3D is a de-novo designed three-helix bundle protein. Like most naturally occurring helical proteins, it is mechanically labile with an unfolding force of <15pN, revealed by atomic force microscopy-based single-molecule...

Quantification of carboxylate‐bridged di‐zinc site stability in protein due ferri by single‐molecule force spectroscopy

“…Protein secondary structure is the dominant factor for protein mechanical stability (Crampton & Brockwell, 2010). It becomes clear that this rule applies to naturally occurring and de novo ‐designed proteins (Carrion‐Vazquez et al, 2000; Chakraborty et al, 2011; Wang et al, 2021; Wang, Zhao, et al, 2022).…”

Single-Molecule Force Spectroscopy Reveals the Dynamic HgS Coordination Site in the De Novo-Designed Metalloprotein α3DIV

“…Notably, the strength of several other metal–thiolate bonds in protein system have been measured showing a smaller force than the Hg–S bond. The rupture force of Zn–S bonds in α 3 DIV is ∼160 pN, and that of Fe­(III)–S bonds in rubredoxin is ∼210 pN. , …”

“…The rupture force of Zn−S bonds in α 3 DIV is ∼160 pN, and that of Fe(III)−S bonds in rubredoxin is ∼210 pN. 29,32 De novo protein design is a powerful strategy to understand and expand our knowledge of metalloprotein structure and function. α 3 D/α 3 DIV is one of the first de novo-designed single-peptide-chain proteins with a three-helix-bundle-structured native fold.…”

“…Similarly, the rupture of the metal–ligand bond in a metal-binding protein also leads to a unique ΔLc signal, which can be used to identify the metal–ligand bond. , Thus, several metal-binding proteins have been successfully measured by AFM-SMFS, showing a clear metal–ligand bond rupture event. − Herein, by using AFM-SMFS to study one Hg-α 3 DIV molecule at a time, the dynamic Hg–S coordination condition in α 3 DIV is revealed.…”

One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy

“…[22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] In the AFM-SMFS experiment of proteins, the protein of interest (POI) is immobilized and stretched from two points between the AFM tip and the proteindeposited surface, revealing a wealth of information of protein (un)folding, stability, and interactions. [37][38][39][40][41][42][43][44][45][46] Thus, new methods generating site-specific and strong protein immobilization can be invaluable for obtaining reliable and efficient single-molecule results. [47][48][49][50][51][52] We demonstrated these two methods by immobilizing eGFP and studied its unfolding process by AFM-SMFS, both showing ideal results.…”