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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