title: The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation.
abstract: 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.
authors.affiliation: Department of Biosciences, Rice University , MS-140, 6100 Main Street, Houston, Texas 77005, United States.
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Jones
authors.affiliation: Medical Scientist Training Program, Northwestern University , 303 East Chicago Avenue, Morton 1-670, Chicago, Illinois 60611, United States.
authors.forename: Manan M
authors.initials: MM
authors.lastname: Mehta
authors.affiliation: Department of Biosciences, Rice University , MS-140, 6100 Main Street, Houston, Texas 77005, United States.
authors.forename: Emily E
authors.initials: EE
authors.lastname: Thomas
authors.affiliation: Systems, Synthetic, and Physical Biology Graduate Program, Rice University , 6100 Main MS-180, Houston, Texas 77005, United States.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Atkinson
authors.affiliation: Department of Biological Engineering, Synthetic Biology Center, Massachusetts Institute of Technology , 500 Technology Square, NE47-257, Cambridge, Massachusetts 02139, United States.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Segall-Shapiro
authors.affiliation: Department of Biosciences, Rice University , MS-140, 6100 Main Street, Houston, Texas 77005, United States.
authors.forename: Shirley
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Biosciences, Rice University , MS-140, 6100 Main Street, Houston, Texas 77005, United States.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Silberg
mesh_terms: D000263:Adenylate Kinase / Q000737:chemistry* / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D015723:Gene Library; D016296:Mutagenesis / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods; D019895:Transposases / Q000737:chemistry / Q000235:genetics
chemical_list: D000596:Amino Acids; D004251:DNA Transposable Elements; D010455:Peptides; D011506:Proteins; D010770:Phosphotransferases; D000263:Adenylate Kinase; D019895:Transposases