title: Effect of temperature on the conformation of natively unfolded protein 4E-BP1 in aqueous and mixed solutions containing trifluoroethanol and hexafluoroisopropanol.
abstract: Natively unfolded (intrinsically disordered) proteins have attracted growing attention due to their high abundance in nature, involvement in various signalling and regulatory pathways and direct association with many diseases. In the present work the combined effect of temperature and alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), on the natively unfolded 4E-BP1 protein was studied to elucidate the balance between temperature-induced folding and unfolding in intrinsically disordered proteins. It was shown that elevated temperatures induce reversible partial folding of 4E-BP1 both in buffer and in the mixed solutions containing denaturants. In the mixed solutions containing TFE (HFIP) 4E-BP1 adopts a partially folded helical conformation. As the temperature increases, the initial temperature-induced protein folding is replaced by irreversible unfolding/melting only after a certain level of the protein helicity has been reached. Onset unfolding temperature decreases with TFE (HFIP) concentration in solution. It was shown that an increase in the temperature induces two divergent processes in a natively unfolded protein--hydrophobicity-driven folding and unfolding. Balance between these two processes determines thermal behaviour of a protein. The correlation between heat-induced protein unfolding and the amount of helical content in a protein is revealed. Heat-induced secondary structure formation can be a valuable test to characterise minor changes in the conformations of natively unfolded proteins as a result of site-directed mutagenesis. Mutants with an increased propensity to fold into a structured form reveal different temperature behaviour.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, M60 1QD, UK, el.hackl@gmail.com.
authors.forename: Ellen V
authors.initials: EV
authors.lastname: Hackl
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins; D006358:Hot Temperature; D006801:Humans; D020125:Mutation, Missense; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D020005:Propanols / Q000737:chemistry*; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D014270:Trifluoroethanol / Q000737:chemistry*; D006845:Hydrocarbons, Fluorinated
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D020005:Propanols; D014270:Trifluoroethanol; C087000:EIF4EBP1 protein, human; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated