Disordered Motifs and Domains in Cell Control - October 11-15, 2014

Disordered Motifs and Domains in Cell Control

Poster Session II

48-POS Board 24 Molecular Crowding Stabilizes both the Intrinsically Disordered Calcium-free State and the Folded Calcium-bound State of an RTX Protein: Implication for PROTEIN Secretion Ana-Cristina Sotomayor-Pérez, Orso Subrini, Audrey Hessel, Daniel Ladant, Alexandre Chenal . Department of Structural Biology and Chemistry, CNRS, Institut Pasteur, Paris, France Ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered domains. Here, we present data on an RTX (« Repeat in ToXin ») protein, RC L , an IDP that folds upon calcium binding. RTX motifs are calcium-binding nonapeptide sequences that are found in more than 250 virulence factors secreted by Gram-negative pathogenic bacteria. Using a combination of biophysical approaches, we showed that RC L exhibits the hallmarks of intrinsically disordered proteins in the absence of calcium. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of RC L . Moreover, RC L is an attractive model to investigate the effect of molecular crowding because it offers the opportunity to characterize the crowding effects on the same protein under two drastically distinct folding states. Macromolecular crowding affects most chemical equilibria in living cells by sterically restricting the available space. We showed that the crowding agent Ficoll70 did not affect the structural content of the apo-state and holo-state of RC L but increased the protein affinity for calcium. Besides, Ficoll70 strongly stabilizes both states of RC L , increasing their half-melting temperature (∆Tm), without affecting enthalpy changes. The power law dependence of the Tm increase on the volume fraction allow d the stimation of the Flory exponent of the thermally unfolded states. Altogether, our data suggest that, in the apo-state as found in the crowded bacterial cytosol, RTX proteins adopt extended unfolded conformations that may facilitate protein export by the secretion machinery. Subsequently, calcium gradient across bacterial cell wall and crowding also enhances the calcium-dependent folding and stability of RTX proteins once secreted in the extracellular milieu. Articles on this topic: Sotomayor-Pérez et al., (2013). Journal of the American Chemical Society (F1000)

Toxin Gets Support From A Crowd. (2013). JACS Spotlight Sotomayor-Pérez et al., (2011). Journal of Biological Chemistry Chenal et al., (2010). Biophysical Journal Sotomayor-Pérez et al., (2010). Journal of Molecular Biololgy Chenal et al., (2009). Journal of Biological Chemistry Bauche et al., (2006). Journal of Biological Chemistry Methods: Sotomayor-Pérez et al., in Methods Mol Biol. 2012 Karst et al., in Methods Mol Biol. 2012

Circular Dichroism, Synchrotron Radiation CD, Fourier Transform Infrared Spectroscopy, Nuclear Magnetic Resonance, Fluorescence, Size Exclusion Chromatography followed by UV, RI, Static Light Scattering and Intrinsic Viscosity, Analytical Ultra Centrifugation, Quasi-Elastic Light Scattering, Electrophoretic Mobility, Small-Angle X-ray Scattering.

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