Abstract
The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240–300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein.
Highlights
IntroductionAll biologically significant processes, including folding, binding of small molecules, proteinprotein interactions, protein aggregation, the formation of amyloid fibrils, etc., take place in a cell in the environment crowded with biological macromolecules (Chebotareva, Filippov & Kurganov, 2015; Uversky et al, 2002; Zimmerman & Minton, 1993; Hatters, Minton & Howlett, 2002; Kuznetsova, Turoverov & Uversky, 2014; Kuznetsova et al, 2015; Minton, 2005) and low-molecular-weight organic substances such as metabolites and osmolytes (Sharp, 2015; Bai et al, 2017; Al-Ayoubi et al, 2017)
Testing of the crowding conditions inside living cells using a FRET sensor designed as PEG modified with donor–acceptor pair of ATTO dyes revealed that the excluded volume effect is not the dominant factor influencing the behavior of macromolecules, its effect is enhanced under osmotic stress (Gnutt et al, 2015)
We tested the effect of crowding agents Dextran-40 and Dextran-70 at a concentration of 240 mg/ml (Fig. 1) and PEG-8000 at a concentration of 80, 120 and 300 mg/ml (Fig. 2) on the structure of iRFP713 in the holoform using absorption, fluorescence spectroscopy and circular dichroism
Summary
All biologically significant processes, including folding, binding of small molecules, proteinprotein interactions, protein aggregation, the formation of amyloid fibrils, etc., take place in a cell in the environment crowded with biological macromolecules (Chebotareva, Filippov & Kurganov, 2015; Uversky et al, 2002; Zimmerman & Minton, 1993; Hatters, Minton & Howlett, 2002; Kuznetsova, Turoverov & Uversky, 2014; Kuznetsova et al, 2015; Minton, 2005) and low-molecular-weight organic substances such as metabolites and osmolytes (Sharp, 2015; Bai et al, 2017; Al-Ayoubi et al, 2017). It is generally accepted that the conditions of molecular crowding, under which a part of the cellular space is occupied by biological molecules and becomes unavailable to other molecules (Zimmerman & Minton, 1993; Minton, 2001), affect biological processes due to the excluded volume effect (Cheung, Klimov & Thirumalai, 2005; Minton, 1993; Tokuriki et al, 2004). This conception allows explaining not all experimental data (Mittal, Chowhan & Singh, 2015; Phillip & Schreiber, 2013). Biological macromolecules with different physicochemical properties, for example, different proteins and nucleic acids, can respond differently to changes in the solvent properties of water (Kuznetsova, Turoverov & Uversky, 2014; Mittal, Chowhan & Singh, 2015; Zaslavsky & Uversky, 2018)
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