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  • Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition.

Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition.

Structure (London, England : 1993) (2020-05-04)
Abigail R Lambert, Jazmine P Hallinan, Rachel Werther, Dawid Głów, Barry L Stoddard
要旨

The redesign of a macromolecular binding interface and corresponding alteration of recognition specificity is a challenging endeavor that remains recalcitrant to computational approaches. This is particularly true for the redesign of DNA binding specificity, which is highly dependent upon bending, hydrogen bonds, electrostatic contacts, and the presence of solvent and counterions throughout the molecular interface. Thus, redesign of protein-DNA binding specificity generally requires iterative rounds of amino acid randomization coupled to selections. Here, we describe the importance of scaffold thermostability for protein engineering, coupled with a strategy that exploits the protein's specificity profile, to redesign the specificity of a pair of meganucleases toward three separate genomic targets. We determine and describe a series of changes in protein sequence, stability, structure, and activity that accumulate during the engineering process, culminating in fully retargeted endonucleases.

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製品内容

Sigma-Aldrich
フェニルメチルスルホニルフルオリド, ≥98.5% (GC)
Sigma-Aldrich
フルオロセイン(遊離酸), Dye content 95 %
Sigma-Aldrich
アデニン ヘミ硫酸塩, powder, BioReagent, suitable for cell culture