6sp5

Publication Abstract from PubMed

Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (T m = 73.5 degrees C; DeltaT m > 23 degrees C). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 A and 1.6 A resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the alphabetaalpha-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance ( approximately 27 A) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts.

Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst.,Markova K, Chmelova K, Marques SM, Carpentier P, Bednar D, Damborsky J, Marek M Chem Sci. 2020 Sep 11;11(41):11162-11178. doi: 10.1039/d0sc03367g. PMID:34094357^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.