How general are the determinants of stability and activity? An evolutionary study with perturbed beta-lactamases
Final Report Abstract
All set specific aims of the first and second funding period were successfully reached. At the outset it was often questioned whether a structural‐perturbation and compensation strategy would ever work, since it was counter intuitive to many researchers to destabilize a protein to gain stability. We not only demonstrated that the idea worked as predicted but we also provide detailed analyses of every step to deepen our knowledge on the evolution of enzyme stability. We used the enzyme TEM ß‐lactamase as a model, because it is well studied and has a potential application in prodrug activation therapy. Aspects of trait in linkage in protein stability against various types of stress have been observed in previous stability studies. However, to our knowledge we provide the first thorough analysis with various types of stress ranging from structural perturbation and proteases and heat to organic solvents demonstrating the scope and exploitation of the stability‐trait linkage in proteins. In addition our approach with serial truncations is a first step towards the minimization of protein structures which previously have been defined as immutable. These findings have major implications for future research since this new concept enables researchers in academia and industry to design novel stabilization strategies. The previously prevalent view of the "first law of Evolution" which states that "you get what you screen for" would mean that specific screening for each stability trait is required. Thus, it was attempted to select in thermophilic organisms or to screen in organic solvents. With our smart destabilization‐compensation strategy we can select under the typical working conditions of an enzyme and due to the orthogonality of the obtained mutations and the perturbation we can revoke the perturbation and obtain added stability of the whole enzyme as well as several other desired stability traits. Since we compare stepwise truncation and destabilization of an enzyme core we gain insight in how stability can be obtained via various routes. Furthermore, we provide an improved lactamase for the protein‐fragment complementation assay which be benchmarked with interaction partners of known affinity. Importantly, we also developed a novel combination of protein‐fragment complementation assay in combination with a TAT‐pathway translocation selection strategy.
