Accession Number : ADA509891


Title :   A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain from Bordetella pertussis Adenylate Cyclase


Descriptive Note : Journal article


Corporate Author : COLUMBIA UNIV NEW YORK DEPT OF CHEMICAL ENGINEERING


Personal Author(s) : Szilvay, Geza R. ; Blenner, Mark A. ; Shur, Oren ; Cropek, Donald M. ; Banta, Scott


Full Text : http://www.dtic.mil/get-tr-doc/pdf?AD=ADA509891


Report Date : 22 OCT 2009


Pagination or Media Count : 11


Abstract : 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 ? roll structure. Here we present an in vitro Foerster 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.


Descriptors :   *BORDETELLA PERTUSSIS , *TOXINS AND ANTITOXINS , *CALCIUM , BIOLOGY , SPECTROSCOPY , PROTEINS , ORDER DISORDER TRANSFORMATIONS , CIRCULAR , DICHROISM , AMINO ACIDS , TRIGGER CIRCUITS , TRYPTOPHAN , ADENYL CYCLASE , FLUORESCENCE , IONIC STRENGTH , FUNCTIONS , IONS


Subject Categories : BIOCHEMISRTY
      TOXICOLOGY
      MICROBIOLOGY


Distribution Statement : APPROVED FOR PUBLIC RELEASE