A chemical chaperone induces inhomogeneous conformational changes in flexible proteins
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Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the conformation of the marginally stable LPPs to avoid their inappropriate protein/protein interactions and aggregation.Keywords:
Osmolyte
Chemical chaperone
Chaperone (clinical)
Methylamines
Protein Stability
Osmolyte
Methylamines
Osmoregulation
Dimethylsulfoniopropionate
Chaotropic agent
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Osmolytes are well known to protect the protein structure against different chemical and physical denaturants.
Osmolyte
Chemical chaperone
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Osmolyte
Chemical chaperone
Macromolecular Crowding
Co-chaperone
Folding (DSP implementation)
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Molecular and chemical chaperones are key components of the two main mechanisms that ensure structural stability and activity under environmental stresses. Yet, chemical chaperones are often regarded only as osmolytes and their role beyond osmotic regulation is not fully understood. Here, we systematically studied a large group of chemical chaperones, representatives of diverse chemical families, for their protective influence under either thermal or chemical stresses. Consistent with previous studies, we observed that in spite of the structural similarity between sugars and sugar alcohols, they have an apparent difference in their protective potential. Our results support the notion that the protective activity is mediated by the solvent and the presence of water is essential. In the current work we revealed that i) polyols and sugars have a completely different profile of protective activity toward trifluoroethanol and thermal stress; ii) minor changes in solvent composition that do not affect enzyme activity, yet have a great effect on the ability of osmolytes to act as protectants and iii) increasing the number of active groups of carbohydrates makes them better protectants while increasing the number of active groups of methylamines does not, as revealed by attempts to synthesize de novo designed methylamines with multiple functional groups.
Osmolyte
Methylamines
Chemical chaperone
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Organic osmolytes are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume. All known osmolytes are amino acids and derivatives, polyols and sugars, methylamines, and urea; unlike salt ions, most are "compatible," i.e., do not perturb macromolecules. In addition, some stabilize macromolecules and are "counteracting" towards perturbants, e.g., methylamines can stabilize proteins and ligand binding against perturbations by urea in elasmobranchs and mammalian kidney, and (our latest findings) high hydrostatic pressure in deep-sea animals. Methylamines appear to coordinate water molecules tightly, resulting in osmolyte exclusion from hydration layers of peptide backbones. This makes unfolded protein conformations entropically unfavorable (work of Timasheff, Galinski, Bolen and coworkers). These properties have led to proposed uses in biotechnology, agriculture and medicine, including improved biochemical methods, in vitro rescue of misfolded proteins in cystic fibrosis and prion diseases (work of Welch and others), and plants engineered for drought and salt tolerance. These properties also explain some but not all of the considerable variation in osmolyte composition among species with different metabolisms and habitats, and among and within mammalian tissues in development.
Osmolyte
Methylamines
Osmoregulation
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Organic osmolytes are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume. All known osmolytes are amino acids and derivatives, polyols and sugars, methylamines, and urea; unlike salt ions, most are "compatible," i.e., do not perturb macromolecules. In addition, some stabilize macromolecules and are "counteracting" towards perturbants, e.g., methylamines can stabilize proteins and ligand binding against perturbations by urea in elasmobranchs and mammalian kidney, and (our latest findings) high hydrostatic pressure in deep-sea animals. Methylamines appear to coordinate water molecules tightly, resulting in osmolyte exclusion from hydration layers of peptide backbones. This makes unfolded protein conformations entropically unfavorable (work of Timasheff, Galinski, Bolen and coworkers). These properties have led to proposed uses in biotechnology, agriculture and medicine, including improved biochemical methods, in vitro rescue of misfolded proteins in cystic fibrosis and prion diseases (work of Welch and others), and plants engineered for drought and salt tolerance. These properties also explain some but not all of the considerable variation in osmolyte composition among species with different metabolisms and habitats, and among and within mammalian tissues in development.
Osmolyte
Methylamines
Osmoregulation
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Citations (74)
Osmolyte
Denaturation (fissile materials)
Protein Stability
Folding (DSP implementation)
Chemical chaperone
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Osmolyte
Methylamines
Chemical chaperone
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Citations (17)
Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the conformation of the marginally stable LPPs to avoid their inappropriate protein/protein interactions and aggregation.
Osmolyte
Chemical chaperone
Chaperone (clinical)
Methylamines
Protein Stability
Cite
Citations (11)
Osmolyte
Methylamines
Osmoregulation
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