Kokkonen P, Sykora J, Prokop Z, Ghose A, Bednar D, Amaro M, Beerens K, Bidmanova S, Slanska M, Brezovsky J, Damborsky J, Hof M, 2018: Molecular Gating of an Engineered Enzyme Captured in Real Time. Journal of the American Chemical Society (just accepted), doi: 10.1021/jacs.8b09848. full text

Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, a single-molecule fluorescence spectroscopy and molecular dynamics. Photoinduced electron-transfer – fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at single molecule level with the rate constants (k_on = 1822 s^-1, k_off = 60 s^-1) corresponding well with those from the pre-steady-state kinetics (k_-1 = 1100 s^-1, k₁ = 20 s^-1).

Beerens K, Mazurenko S, Kunka A, Marques SM, Hansen N, Musil M, Chaloupkova R, Waterman J, Brezovsky J, Bednar D, Prokop Z, Damborsky J, 2018: Evolutionary Analysis is a Powerful Complement to Energy Calculations for Protein Stabilization. ACS Catalysis 8: 9420-9428. full text

Stability is one of the most important characteristics of proteins employed as biocatalysts, biotherapeutics, and biomaterials, and the role of computational approaches in modifying protein stability is rapidly expanding. We have recently identified stabilizing mutations in haloalkane dehalogenase DhaA using phylogenetic analysis but were not able to reproduce the effects of these mutations using force-field calculations. Here we tested four different hypotheses to explain the molecular basis of stabilization using structural, biochemical, biophysical, and computational analyses. We demonstrate that stabilization of DhaA by the mutations identified using the phylogenetic analysis is driven by both entropy and enthalpy contributions, in contrast to primarily enthalpy-driven stabilization by mutations designed by the force-field calculations. Comprehensive bioinformatics analysis revealed that more than half (53%) of 1 099 evolution-based stabilizing mutations would be evaluated as destabilizing by force-field calculations. Thermodynamic integration considers both folded and unfolded states and can describe the entropic component of stabilization, yet it is not suitable for predictive purposes due to its high computational demands. Altogether, our results strongly suggest that energetic calculations should be complemented by a phylogenetic analysis in protein-stabilization endeavors.

Jurcik A, Bednar D, Byska J, Marques SM, Furmanova K, Daniel L, Kokkonen P, Brezovsky J, Strnad O, Stourac J, Pavelka A, Manak M, Damborsky J, Kozlikova B, 2018: CAVER Analyst 2.0: Analysis and Visualization of Channels and Tunnels in Protein Structures and Molecular Dynamics Trajectories. Bioinformatics (just accepted): doi:10.1093/bioinformatics/bty386. full text

Kaushik S, Marques SM, Khirsariya P, Paruch K, Libichova L,  Brezovsky J, Prokop Z, Chaloupkova R, Damborsky J, 2018: Impact of the Access Tunnel Engineering on Catalysis is Strictly Ligand-Specific. FEBS Journal (just accepted): doi: 10.1111/febs.14418. full text

The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes’ catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products. Thus, we have systematically studied the effects of single-point mutations in a tunnel-lining residue of a haloalkane dehalogenase on the binding kinetics and catalytic conversion of both linear and branched haloalkanes. The hotspot residue Y176 was identified using computer simulations and randomized through saturation mutagenesis, and the resulting variants were screened for shifts in binding rates. Strikingly, opposite effects of the substituted residues on the catalytic efficiency towards linear and branched substrates were observed, which was found to be due to substrate-specific requirements in the critical steps of the respective catalytic cycles. We conclude that not only the catalytic sites but also the access pathways must be tailored specifically for each individual ligand, which is a new paradigm in protein engineering and de novo protein design. A rational approach is proposed here to address more effectively the task of designing ligand-specific tunnels using computational tools.