Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase.
|Title||Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase.|
|Publication Type||Journal Article|
|Year of Publication||2001|
|Authors||Trejo, F, Gelpí Josep-Lluis, Ferrer A, Boronat A, Busquets M, and Cortés A|
|Date Published||2001 Nov|
|Keywords||Catalysis, Cytosol, Guanidine, Kinetics, Malate Dehydrogenase, Models, Molecular, Mutagenesis, Mutation, NAD, Protein Conformation, Protein Engineering, Protein Folding, Recombinant Proteins, Salts, Site-Directed, Temperature, Thermodynamics, Thermus|
Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57-K168 and intrasubunit R149-E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149-E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15 degrees C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.