Schenkmayerova_2020_ChemRxiv__

Reference

Title : Engineering Protein Dynamics of Ancestral Luciferase - Schenkmayerova_2020_Chemrxiv__
Author(s) : Schenkmayerova A , Pinto GP , Toul M , Marek M , Hernychova L , Planas-Iglesias J , Liskova V , Pluskal D , Vasina M , Emond S , Dorr M , Chaloupkova R , Bednar D , Prokop Z , Hollfelder F , Bornscheuer UT , Damborsky J
Ref : Chemrxiv , : , 2020
Abstract :

Insertion-deletion mutations are sources of major functional innovations in naturally evolved proteins, but directed evolution methods rely primarily on substitutions. Here, we report a powerful strategy for engineering backbone dynamics based on InDel mutagenesis of a stable and evolvable template, and its validation in application to a thermostable ancestor of haloalkane dehalogenase and Renilla luciferase. First, extensive multidisciplinary analysis linked the conformational flexibility of a loop-helix fragment to binding of the bulky substrate coelenterazine. The fragment's key role in extant Renilla luciferase was confirmed by transplanting it into the ancestor. This increased its catalytic efficiency 7,000-fold, and fragment-containing mutants showed highly stable glow-type bioluminescence with 100-fold longer half-lives than the flash-type Renilla luciferase RLuc8, thereby addressing a limitation of a popular molecular probe. Thus, our three-step approach: (i) constructing a robust template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of a dynamic feature, provides a potent strategy for discovering protein modifications with globally disruptive but functionally innovative effects.

PubMedSearch : Schenkmayerova_2020_Chemrxiv__
PubMedID:
Gene_locus related to this paper: renre-luc

Related information

Inhibitor Coelenteramide
Gene_locus renre-luc
Structure 6YN2    6S6E    6S97

Citations formats

Schenkmayerova A, Pinto GP, Toul M, Marek M, Hernychova L, Planas-Iglesias J, Liskova V, Pluskal D, Vasina M, Emond S, Dorr M, Chaloupkova R, Bednar D, Prokop Z, Hollfelder F, Bornscheuer UT, Damborsky J (2020)
Engineering Protein Dynamics of Ancestral Luciferase
Chemrxiv :

Schenkmayerova A, Pinto GP, Toul M, Marek M, Hernychova L, Planas-Iglesias J, Liskova V, Pluskal D, Vasina M, Emond S, Dorr M, Chaloupkova R, Bednar D, Prokop Z, Hollfelder F, Bornscheuer UT, Damborsky J (2020)
Chemrxiv :