Unfolding of Cytochromecupon Interaction with Azobenzene-Modified Copolymers

Abstract

Hydrophilic 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 <4M). However, the presence of polymers facilitated unfolding, which occurred at urea concentrations lowered by 2-4 M as compared to unfolding in the absence of polymers (polymer/cyt c ratio of 1:1 g/g). The predominant contribution of coulombic interactions was shown by both the lack of significant impact of the amount of (neutral) azobenzene moieties in the copolymers and the disappearance of destabilization at ionic strength higher than 150 mM. In addition, stability was similar to that of an isolated cyt c, in the presence of a neutral chain bearing acryloyl(oligoethyleneoxide) units instead of the ionized sodium acrylate moieties. DSC measurements showed that in the presence of polymers, cyt c is thermally unfolded in aqueous buffer at temperatures lowered by >20 °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 remote control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarity.

Selective Uptake and Refolding of Globular Proteins in Coacervate Microdroplets

“…These values were increased respectively to +64 ± 22 kJ mol –1 and 5.8 M in the coacervate bulk phase, indicating that the PDDA/PAA matrix stabilized the folded states of BSA. Complexation of negatively charged polyelectrolytes with globular proteins undergoing urea-induced unfolding has been shown to destabilize the structured conformers as a result of competition between polymer/protein intermolecular associations and intraprotein interactions. , Thus, the enhanced stability of the folded conformers observed in the coacervate phase was attributed to the crowded environment experienced by the BSA molecules, which presumably disfavors protein unfolding.…”

Structural transformations of cytochrome c upon interaction with cardiolipin

“…In addition to common denaturants (acid, urea, and GuHCl), many other additives destabilize the native structure of cyt c . Detergent micelles (Bertini et al, 2004; Naeem and Khan, 2004; Bhuyan, 2010), anionic polymers (Antalík et al, 2003; Sun et al, 2012), alcohols (Naeem and Khan, 2004; Bágeľová et al, 2008; Singh et al, 2011), lipid membranes (Pinheiro et al, 1997), fatty acids (Patriarca et al, 2009), many aliphatic anions (Ibanez and Herskovits, 1976), and even low ionic strength (Banci et al, 1998) or added ATP (Antalík and Bágeľová, 1995; Snider et al, 2013) alter structure and/or stability of the protein.…”

Refolding of Aggregation-Prone ScFv Antibody Fragments Assisted by Hydrophobically Modified Poly(sodium acrylate) Derivatives

“…This result is somewhat surprising. Usually, complexation of globular proteins with amphiphilic polymers or surfactants that render the proteins more soluble also tend to facilitate their unfolding . Intermolecular hydrophobic association competes with intraprotein interactions and should destabilize folded states.…”

“…Usually, complexation of globular proteins with amphiphilic polymers or surfactants that render the proteins more soluble also tend to facilitate their unfolding. [49,[52][53][54][55] Intermolecular hydrophobic association competes with intraprotein interactions and should destabilize folded states. Our observation that hydrophobically modified polymers favor more structured scFv suggests a preferential association of the polymer with folded or partly folded conformers that exhibit more hydrophobic surfaces than the completely unfolded states.…”