Examining how adenylate kinase orthologs differ in their tolerance to circular permutation
Jones, Alicia Michelle
Silberg, Jonathan J
Doctor of Philosophy
In nature, protein sequence rearrangements can arise as proteins evolve through a process called circular permutation (CP). These rearrangements result in the covalent linkage of the original protein termini and the creation of new termini elsewhere in the protein backbone. Although the overall tertiary structure of a protein remains the same, CP can have a wide variety of effects on protein dynamics, stability, and activity. The use of CP in protein engineering has yielded proteins with improved catalytic activity, and it has also been valuable in building molecular switches using domain insertion of circularly permuted proteins. However, we cannot yet anticipate how proteins tolerate permutation, and current models are limited in their predictions. My thesis research investigates how adenylate kinase (AK), a well-studied phosphotransferase, tolerates CP. Using AK orthologs with a range of thermostabilities, I improved upon a method for creating combinatorial libraries of circularly permuted AKs using transposase mutagenesis. Application of this method to a hyperthermophilic AK revealed that several structural metrics correlated with permutation tolerance, including: (i) the distance of the new protein termini to the catalytic site, (ii) the sequence diversity at the new termini within a multiple sequence alignment of bacterial AKs, and (iii) the structural deviation of the new termini in superimposed AK structures. In addition, I showed that a trade-off exists between consistently expressing permuted AK in a combinatorial library and minimizing N-terminal peptide additions. Subsequent studies explored AK tolerance to permutation in thermophilic, mesophilic, and psychrophilic species. Next generation sequencing was used to assess biases in permutation libraries and to mine these permuted libraries for functional AK. The method described for building combinatorial libraries using transposase mutagenesis will be applicable to any protein and will simplify studies of permutation tolerance across many proteins in parallel. The results of my thesis work also have implications for understanding protein tolerance to CP by providing insight into the structural parameters that correlate with retention of structure and function. Finally, comparisons of AK permutation tolerance in multiple orthologs will aid in the development of better models for predicting protein tolerance to permutation.