Catch and Release: Engineered Allosterically Regulated β-Roll Peptides Enable On/Off Biomolecular Recognition

Beyza Bulutoglu, Kevin Dooley, Géza Szilvay, Mark Blenner, Scott Banta (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

10 Citations (Scopus)

Abstract

Alternative scaffolds for biomolecular recognition are being developed to overcome some of the limitations associated with immunoglobulin domains. The repeat-in-toxin (RTX) domain is a repeat protein sequence that reversibly adopts the β-roll secondary structure motif specifically upon calcium binding. This conformational change was exploited for controlled biomolecular recognition. Using ribosome display, an RTX peptide library was selected to identify binders to a model protein, lysozyme, exclusively in the folded state of the peptide. Several mutants were identified with low micromolar dissociation constants. After concatenation of the mutants, a 500-fold increase in the overall affinity for lysozyme was achieved leading to a peptide with an apparent dissociation constant of 65 nM. This mutant was immobilized for affinity chromatography experiments, and the on/off nature of the molecular recognition was demonstrated as the target is captured from a mixture in the presence of calcium and is released in the absence of calcium as the RTX peptides lose their β-roll structure. This work presents the design of a new stimulus-responsive scaffold that can be used for environmentally responsive specific molecular recognition and self-assembly.

Original languageEnglish
Pages (from-to)1732-1741
Number of pages10
JournalACS Synthetic Biology
Volume6
Issue number9
DOIs
Publication statusPublished - 15 Sep 2017
MoE publication typeA1 Journal article-refereed

Keywords

  • conditional target binding
  • disordered-to-ordered transition
  • molecular recognition
  • protein switch
  • RTX domain

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