Thermostability of Enzymes from Molecular Dynamics Simulations

Abstract

Thermodynamic stability is a central requirement for protein function, and one goal of protein engineering is improvement of stability, particularly for applications in biotechnology. Herein, molecular dynamics simulations are used to predict in vitro thermostability of members of the bacterial ribonuclease HI (RNase H) family of endonucleases. The temperature dependence of the generalized order parameter, S, for four RNase H homologues, from psychrotrophic, mesophilic, and thermophilic organisms, is highly correlated with experimentally determined melting temperatures and with calculated free energies of folding at the midpoint temperature of the simulations. This study provides an approach for in silico mutational screens to improve thermostability of biologically and industrially relevant enzymes.

Figure 1

Λ and R values for ecRNH (black), ctRNH (green), soRNH (blue), ttRNH (red). Prolines do not have an NH bond vector and were omitted from the sequences for this figure. Green and red bars represent sheets and helices, respectively.

Figure 2

Histograms of Λ values for the four organisms with experimentally determined T_m values: soRNH (blue), ctRNH (green), ecRNH (black), ttRNH (red). The inset shows the homomorphic relationship between average Λ values and experimentally determined T_m.

Figure 3

Structures of soRNH (left) and ttRNH (right). soRNH: Residues with Λ values over 1.2 in green, residues with Λ values over 2 in blue. ttRNH: Residues with Λ values under 0.5 in orange, residues with Λ values under 0.3 in red. The iG80b in ttRNH insertion is shown as purple spheres.