The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation

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dc.citation.firstpage415en_US
dc.citation.issueNumber5en_US
dc.citation.journalTitleACS Synthetic Biologyen_US
dc.citation.lastpage425en_US
dc.citation.volumeNumber5en_US
dc.contributor.authorJones, Alicia M.en_US
dc.contributor.authorMehta, Manan M.en_US
dc.contributor.authorThomas, Emily E.en_US
dc.contributor.authorAtkinson, Joshua T.en_US
dc.contributor.authorSegall-Shapiro, Thomas H.en_US
dc.contributor.authorLiu, Shirleyen_US
dc.contributor.authorSilberg, Jonathan J.en_US
dc.contributor.orgSystems, Synthetic, and Physical Biology Programen_US
dc.date.accessioned2017-05-03T16:12:49Zen_US
dc.date.available2017-05-03T16:12:49Zen_US
dc.date.issued2016en_US
dc.description.abstractProteins 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.en_US
dc.identifier.citationJones, Alicia M., Mehta, Manan M., Thomas, Emily E., et al.. "The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation." <i>ACS Synthetic Biology,</i> 5, no. 5 (2016) American Chemical Society: 415-425. https://doi.org/10.1021/acssynbio.5b00305.en_US
dc.identifier.doihttps://doi.org/10.1021/acssynbio.5b00305en_US
dc.identifier.urihttps://hdl.handle.net/1911/94126en_US
dc.language.isoengen_US
dc.publisherAmerican Chemical Societyen_US
dc.rightsThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the American Chemical Society.en_US
dc.titleThe Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutationen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpost-printen_US
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