The structure of a thermophilic kinase shapes fitness upon random circular permutation
Autor: | Shirley Liu, Manan M. Mehta, Emily E. Thomas, Joshua T. Atkinson, Jonathan J. Silberg, Alicia M. Jones, Thomas H. Segall-Shapiro |
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Jazyk: | angličtina |
Rok vydání: | 2016 |
Předmět: |
0301 basic medicine
Transposable element Biomedical Engineering Mutagenesis (molecular biology technique) Transposases Biology Cleavage (embryo) Protein Engineering Biochemistry Genetics and Molecular Biology (miscellaneous) Article Protein Structure Secondary 03 medical and health sciences Protein structure Amino Acids Transposase Sequence (medicine) Gene Library Genetics 030102 biochemistry & molecular biology Adenylate Kinase Phosphotransferases Proteins General Medicine Protein engineering Circular permutation in proteins 030104 developmental biology Mutagenesis DNA Transposable Elements Synthetic Biology Peptides |
Popis: | Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement in which native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein's functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AKs with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and it reveals a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection. |
Databáze: | OpenAIRE |
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