title: Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants.
abstract: Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant-robust broad-specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS-PAGE and N-terminal sequencing. We observe well-defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for alpha-lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates.
authors.affiliation: Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, Aalborg DK-9000, Denmark.
authors.forename: Jonas Høeg
authors.initials: JH
authors.lastname: Hansen
authors.forename: Steen Vang
authors.initials: SV
authors.lastname: Petersen
authors.forename: Kell Kleiner
authors.initials: KK
authors.lastname: Andersen
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.forename: Ture
authors.initials: T
authors.lastname: Damhus
authors.forename: Daniel
authors.initials: D
authors.lastname: Otzen
mesh_terms: D000818:Animals; D002417:Cattle; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology*; D013696:Temperature
chemical_list: D011506:Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D010447:Peptide Hydrolases