Engineering Enzyme Stability and Resistance to an Organic Cosolvent by Modification of Residues in the Access Tunnel

Authors


  • Dr. Jiri Jarkovsky (Institute of Biostatistics and Analysis, Masaryk University, Brno) is gratefully acknowledged for help with statistical data analysis. T.K. acknowledges scholarships from the Federation of European Microbiological Societies and Ernst-Moritz-Arndt-University for research visits to the Department of Biotechnology and Enzyme Catalysis, Greifswald (Germany). This work was supported by the Ministry of Education of the Czech Republic (LC06010 and MSM0021622412), the Grant Agency of the Czech Republic (203/08/0114, P202/10/1435, and P207/12/0775), the Grant Agency of the Czech Academy of Sciences (IAA401630901) and the European Regional Development Fund (CZ.1.05/2.1.00/01.0001). U.T.B. thanks the German Research Foundation (DFG) for financial support (SPP 1170, Bo1864/4-1). MetaCentrum is acknowledged for providing access to computing facilities, supported by the Czech Ministry of Education of the Czech Republic (LM2010005).

Abstract

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Mutations targeting as few as four residues lining the access tunnel extended the half-life of an enzyme in 40 % dimethyl sulfoxide from minutes to weeks and increased its melting temperature by 19 °C. Protein crystallography and molecular dynamics revealed that the tunnel residue packing is a key determinant of protein stability and the active-site accessibility for cosolvent molecules (red dots).

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