Careful balance between structural stability and flexibility is a hallmark of enzymatic function, and temperature can affect both properties. Canonical (fixed-backbone) enzyme design strategies currently do not consider the role of these properties. Herein, we describe the rational design of 100 temperature-adapted adenylate kinase enzymes using a multistate design strategy that incorporates the impact of conformational changes to backbone structure and stability, in addition to experimental analysis of thermostability and function. Comparison of the experimental temperature of maximum activity to the melting temperature across all 100 variants reveals a strong correlation between these two parameters. In turn, experimental stability data were used to produce accurate predictions of thermostability, providing the requisite complement for de novo temperature-adapted enzyme design. In principle, this level of design-based analysis can be applied to any protein, paving the way toward identifying and understanding the hallmarks of the thermodynamic and structural limits of function.
Submitter: Barry Olafson
Submission Date: Oct. 30, 2016, 4:59 p.m.
|Number of data points||1283|
|Assays/Quantities/Protocols||Experimental Assay: ΔG(T*) ; Experimental Assay: ΔCp ; Experimental Assay: T* ; Experimental Assay: Tc ; Experimental Assay: ΔHm ; Experimental Assay: Tma ; Experimental Assay: Tm ; Derived Quantity: SD of Tm ; Derived Quantity: SD of ΔCp ; Derived Quantity: SD of Tma ; Derived Quantity: ΔSm ; Derived Quantity: SD of ΔHm|
|Libraries||Thermodynamic and thermoactivity parameters of AdK designs|