(545e) Engineering Tools for Analysis of Intrinsically Disordered Proteins: Entropic Stabilization Enables a Type I Secretion Calcium Switch

Intrinsically disordered proteins are involved in myriad biological processes. These proteins have long been overlooked because they lack the secondary and tertiary structure normally associated with biological function. Many of these proteins, however, can form secondary structures upon interaction with associated ligands. These specialized properties allow intrinsically disordered proteins to play important roles in biological decisions, and as such are prevalent in transcriptional regulation as well signal transduction pathways. While many programs exist that can predict intrinsic disorder in proteins, there are not many well developed tools available to analyze structural changes for this increasingly important class of proteins. We present work towards the characterization of a beta-roll forming Repeat in Toxin (RTX) domain from the adenlyate cyclase toxin of Bordetella pertussis. The calcium-induced structural changes of this domain have been studied using traditional methods, such as circular dichroism spectroscopy, tryptophan fluorescence and bis-ANS fluorescence. These results are compared with Forster Resonance Energy Transfer (FRET) measurements of the RTX domain. Our results indicate the importance of flanking groups for the calcium-induced folding, or the disorder/order transition. The RTX domain is part of the secretion signal associated with Type I Secretion Systems (TISS). The RTX domain acts as a calcium switch that helps to keep the adenylate cyclase toxin unfolded, and thus competent for secretion. We show that the only the C-terminal flanking group provides the necessary stabilization to permit calcium-induced structural formation. Further, we have shown that several different proteins endow similar responsiveness to the RTX domain. This generality indicates the dominance of an entropic capping stabilization at the C-terminal end. This supports the notion that T1SS passengers are secreted and folded from the C-terminal end.