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Investigating mechanical properties of a protein by single molecule atomic force microscopy Shen, Tao

Abstract

Single molecule AFM is a powerful technique affording the opportunity to understand the mechanical properties of proteins at the level of a single molecule. In Combining with the protein engineering techniques, single molecule allows us to understand protein folding/unfolding mechanisms and develop methods to tune the mechanical stability. Here, we use a small protein, GB1, the B1 IgG binding domain of protein G from Streptococcus, as a model system. In this thesis, we employed bi-His metal binding sites to probe the mechanical unfolding transition state of GB1 and rationally enhance the mechanical stability of GB1 mutant, G6-53. The transition state cannot be trapped and detected by the usual structural methods because of its high free energy. It remains a challenging task and research focus. In Chapter 3, we directly probed the mechanical unfolding transition state structure of protein GB1. The results demonstrate that the contacts between the force-bearing strands 1 and 4 are largely disrupted at the transition state, whereas the first β-hairpin and α-helix were largely intact. The second hairpin was partially disrupted. These results are in close agreement with, and provide a benchmark for, MD simulations. The mechanical stability is critical for the overall mechanical properties of elastomeric proteins. Elastomeric proteins provide tissues with extensibility, elasticity, and mechanical strength. In Chapter 4, we enhanced the mechanical stability of G6-53 with different metal ions. We demonstrated that all four divalent metal ions, Ni²⁺, Co²⁺, Zn²⁺ and Cu²⁺, enhance the mechanical stability of G6-53 to different degrees. Because this process is completely reversible, the protein can be treated like a switch. Moreover, the resultant unfolding force difference between Co²⁺ and Zn²⁺ or Zn²⁺ and Cu²⁺ is ~ 20 pN. Thus, various metal ions can be used to fine tune the mechanical stability of proteins.

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