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Active TEM‐1 β‐lactamase mutants with random peptides inserted in three contiguous surface loops
Author(s) -
Mathonet Pascale,
Deherve Julie,
Soumillion Patrice,
Fastrez Jacques
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.062303606
Subject(s) - active site , mutagenesis , mutant , protein engineering , cysteine , amino acid , directed evolution , site directed mutagenesis , binding site , saturated mutagenesis , biophysics , stereochemistry , chemistry , biochemistry , enzyme , biology , gene
Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non‐natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM‐1 β‐lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two β‐strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well‐conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N‐terminal helix and the first β‐strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final β‐strand to the C‐terminal helix, particularly if the wild‐type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium‐sized loops. The combination of these activity‐selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.