| To investigate connections between structure, function and dynamics of proteins, I studied adenylate kinases from the psychrophile Bacillus globisporus, the mesophile Bacillus subtilis, and the thermophile Bacillus stearothermophilus. As expected, their catalytic activities and thermal transitions scale with the operating temperatures of the source organisms. The crystal structures of the psychrophilic and mesophilic adenylate kinases have been solved and compared with the previously determined thermophilic structure. The comparative structural analysis suggests modification of electrostatic and/or hydrophobic interactions as the molecular basis of the disparate stabilities. Several residue substitutions were also proposed to be important for the difference, and the proposition was tested by analyzing mutant adenylate kinases produced by site directed mutagenesis. Dynamics of the wild type and mutant enzymes have been studied by performing molecular dynamics simulations. Although it is not possible to find a clear connection between global flexibilities and temperature preferences of the enzymes, the simulations provided insights into identifying and engineering favorable ion pairs for thermal stability. The conclusions from the simulations regarding the ion pairs were consistent with the results from the in vitro mutagenesis. To further investigate the connections between structure, function and dynamics in adenylate kinase, I constructed chimeras from the mesophilic and thermophilic adenylate kinases. The high similarity of the two homologues and a new method using synthetic genes allowed specific regions of the enzymes to be exchanged, producing a series of fully functional chimeric adenylate kinases. Differential scanning calorimetry and activity assays at various temperatures of the chimeras subsequently revealed spatial separation of stability and activity control, indicating specific contributions of dynamics to catalysis in adenylate kinase. |
