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

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dc.citation.firstpage415
dc.citation.issueNumber5
dc.citation.journalTitleACS Synthetic Biology
dc.citation.lastpage425
dc.citation.volumeNumber5
dc.contributor.authorJones, Alicia M.
dc.contributor.authorMehta, Manan M.
dc.contributor.authorThomas, Emily E.
dc.contributor.authorAtkinson, Joshua T.
dc.contributor.authorSegall-Shapiro, Thomas H.
dc.contributor.authorLiu, Shirley
dc.contributor.authorSilberg, Jonathan J.
dc.contributor.orgSystems, Synthetic, and Physical Biology Program
dc.date.accessioned2017-05-03T16:12:49Z
dc.date.available2017-05-03T16:12:49Z
dc.date.issued2016
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.
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.
dc.identifier.doihttps://doi.org/10.1021/acssynbio.5b00305
dc.identifier.urihttps://hdl.handle.net/1911/94126
dc.language.isoeng
dc.publisherAmerican Chemical Society
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.
dc.titleThe Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation
dc.typeJournal article
dc.type.dcmiText
dc.type.publicationpost-print
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