A FRET-based method for probing the conformational behavior of an intrinsically disordered repeat domain from Bordetella pertussis adenylate cyclase

Geza Szilvay, M. A. Blenner, O. Shur, D. M. Cropek, S. Banta (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

36 Citations (Scopus)


A better understanding of the conformational changes exhibited by intrinsically disordered proteins is necessary as we continue to unravel their myriad biological functions. In repeats in toxin (RTX) domains, calcium binding triggers the natively unstructured domain to adopt a β roll structure. Here we present an in vitro Förster resonance energy transfer (FRET)-based method for the investigation of the conformational behavior of an RTX domain from the Bordetella pertussis adenylate cyclase consisting of nine repeat units. Equilibrium and stopped-flow FRET between fluorescent proteins, attached to the termini of the domain, were measured in an analysis of the end-to-end distance changes in the RTX domain. The method was complemented with circular dichroism spectroscopy, tryptophan fluorescence, and bis-ANS dye binding. High ionic strength was observed to decrease the calcium affinity of the RTX domain. A truncation and single amino acid mutations yielded insights into the structural determinants of β roll formation. Mutating the conserved Asp residue in one of the nine repeats significantly reduced the affinity of the domains for calcium ions. Removal of the sequences flanking the repeat domain prevented folding, but replacing them with fluorescent proteins restored the conformational behavior, suggesting an entropic stabilization. The FRET-based method is a useful technique that complements other low-resolution techniques for investigating the dynamic conformational behavior of the RTX domain and other intrinsically disordered protein domains.
Original languageEnglish
Pages (from-to)11273-11282
Number of pages10
Issue number47
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

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