Effects of Calcium and Protons on the Secondary Structure of the Nodulation Protein NodO from Rhizobium leguminosarum biovar viciae

Serra, Mauro Dalla; Sutton, J. Mark; Höper, Frank; Downie, J. Allan; Menestrina, Gianfranco

doi:10.1006/bbrc.1999.1400

Cited by 8 publications

(1 citation statement)

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“…This segment has five domains that are each composed of tandemly repeated nonapeptides called repeats in toxins (RTX), named so since these motifs are often found in secreted bacterial toxins or virulence factors . Domains formed by tandemly arranged repeated RTX motifs are found in secreted proteins from Gram-negative bacteria, such as in hemolysins, lipases, zinc proteases, nodulation proteins, and cyclolysins − . The RTX motifs have a general consensus sequence of GGXGXDXUX, where U denotes a large hydrophobic amino acid and X represents any amino acid.…”

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confidence: 99%

A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain fromBordetella pertussisAdenylate Cyclase

et al. 2009

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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 beta roll structure. Here we present an in vitro Forster 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 beta 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.

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confidence: 99%