Unfolding of Cytochrome c upon Interaction with Azobenzene-Modified Copolymers

International audienceHydrophilic or amphiphilic macromolecules are common organic matrices used to encapsulate and protect fragile drugs such as proteins. Polymer cargoes are in addition designed for remote control of,protein delivery, upon imparting the macromolecules with stimuli-responsive properties, such as light-triggered :polarity switches. The effect of interaction between polymers and proteins on the Stability of the proteins is, however, rarely Investigated. Here we studied the unfolding/folding equilibrium of. cytochrome c.(cyt c) under its oxidized or reduced forms, in the presence of various,amphiphilic copolymers (by circular dichroism and;intrinsic fluorescence measurements), As models of stimuli-responsive amphiphilic chains, we considered Poly(acrylic acid) derivatives, modified to contain hydrophobic,, light-responsive azobenzene moieties.. These copolymers are, thus, capable to develop both ionic (Under their sodium forms at pH > 8) and hydrophobic associations with the basic protein cyt c (isoelectric point of 10.0). In aqueous buffer upon increasing urea concentrations, cyt c underwent unfolding, at [urea] Of 9-10 M, which was analyzed under the framework Of the equilibrium between two States (native unfolded). In the presence of polymers, the native folding of cyt c was preserved at low Concentrations Of urea (typically 20 degrees C as compared to thermal unfolding in the absence of polymers. Upon exposure to UV light, properties of the polymers chains were perturbed in situ, upon cis/trans isomerization of the azobenzene groups. In polymers displaying a photoresponsive polarity and hydrophobicity switch (conventional azobenzene), the stability of cyt c was not affected by the exposure to light. In contrast, when photoionization occurred (using an hydroxyl-azobenzene whose pK(a) can be photoshifted); unfolding was initiated upon exposure to light. Altogether, these results show that coulombic binding is a predominant driving force that facilitates unfolding in water/urea solutions. In regard to the design of light-responsive systems for protein handling and control of folding, we, conclude that control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarit