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The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation
journal contribution
posted on 2016-03-14, 00:00 authored by Alicia
M. Jones, Manan M. Mehta, Emily E. Thomas, Joshua T. Atkinson, Thomas H. Segall-Shapiro, Shirley Liu, Jonathan J. SilbergProteins
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.
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retentionlack cleavage sitescircularly permuted proteinsparameterpeptide additionstolerancepermuted protein librariespermutationthermophilic adenylate kinaseterminifunctionphosphotransfer sitepermuted protein expressionuse transposase mutagenesisvariantThermophilic Kinase Shapes FitnessAK family sequence alignmentRandom Circular Permutation Proteins
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