title: Limited proteolysis of natively unfolded protein 4E-BP1 in the presence of trifluoroethanol.
abstract: Natively unfolded (intrinsically disordered (ID) proteins) have been attracting an increasing attention due to their involvement in many regulatory processes. Natively unfolded proteins can fold upon binding to their metabolic partners. Coupled folding and binding events usually involve only relatively short motifs (binding motifs). These binding motifs which are able to fold should have an increased propensity to form a secondary structure. The aim of the present work was to probe the conformation of the intrinsically disordered protein 4E-BP1 in the native and partly folded states by limited proteolysis and to reveal regions with a high propensity to form an ordered structure. Trifuoroethanol (TFE) in low concentrations (up to 15 vol%) was applied to increase the helical population of protein regions with a high intrinsic propensity to fold. When forming helical structures, these regions lose mobility and become more protected from proteases than random/unfolded protein regions. Limited proteolysis followed by mass spectrometry analysis allows identification of the regions with decreased mobility in TFE solutions. Trypsin and V8 proteases were used to perform limited proteolysis of the 4E-BP1 protein in buffer and in solutions with low TFE concentrations at 37°C and at elevated temperatures (42 and 50°C). Comparison of the results obtained with the previously established 4E-BP1 structure and the binding motif illustrates the ability of limited proteolysis in the presence of a folding assistant (TFE) to map the regions with high and low propensities to form a secondary structure revealing potential binding motifs inside the intrinsically disordered protein.
authors.affiliation: Department of Biomolecular Sciences, Faculty of Life Sciences, The University of Manchester, Manchester M60 1QD, UK.
authors.forename: Ellen V
authors.initials: EV
authors.lastname: Hackl
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D002021:Buffers; D018797:Cell Cycle Proteins; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D013058:Mass Spectrometry; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D020005:Propanols / Q000494:pharmacology; D017433:Protein Structure, Secondary; D058767:Protein Unfolding / Q000187:drug effects*; D059748:Proteolysis / Q000187:drug effects*; D012697:Serine Endopeptidases / Q000378:metabolism; D012996:Solutions; D013696:Temperature; D014270:Trifluoroethanol / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism; D006845:Hydrocarbons, Fluorinated
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002021:Buffers; D018797:Cell Cycle Proteins; D010455:Peptides; D010750:Phosphoproteins; D020005:Propanols; D012697:Serine Endopeptidases; D012996:Solutions; D014270:Trifluoroethanol; D014357:Trypsin; C087000:EIF4EBP1 protein, human; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C028344:glutamyl endopeptidase
keywords: 2,2,2-trifluoroethanol; 4E-BP1 protein; intrinsically disordered proteins; limited proteolysis; protease; protein folding; thermolysin; trypsin