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Transforming de novo protein α3D into a mechanically stable protein by zinc binding
Ziyi Wang1, Jingyuan Nie1, Shengcao Shi1
1State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Centre (ChemBIC), Nanjing University, Nanjing, China. pengz@nju.edu.cn.
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 <15 pN, revealed by atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). This protein has been further designed with a tri-cysteine metal-binding site, named α3DIV, which can bind heavy transition metals. Here, we demonstrate that incorporating such a metal-binding site can transform this mechanically labile protein into a stable one. We show that zinc binds to the tri-cysteine site and increases the unfolding force to ∼160 pN. This force is one order of magnitude higher than that of the apo-protein (<15 pN). Moreover, the unfolding mechanism of Zn-α3DIV indicates the correct zinc binding with the tri-cysteine site, forming three mechanostable Zn-thiolate bonds. Thus, α3DIV could be a potential α-helical structure-based building block for synthesizing biomaterials with tunable mechanical properties.
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