title: Effect of pH on the pore forming activity and conformational stability of ostreolysin, a lipid raft-binding protein from the edible mushroom Pleurotus ostreatus.
abstract: Ostreolysin, a pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is a member of the aegerolysin protein family, a novel group of small acidic proteins found in bacteria, molds, mushrooms, and plants. It binds to lipid rafts and interacts specifically with cholesterol-rich lipid domains. In this study, ostreolysin was classified as a single-domain all-beta-structured protein on the basis of cDNA sequencing. pH-induced and thermally induced unfolding of ostreolysin was studied by means of CD, UV absorption, and intrinsic tryptophan fluorescence to characterize conformational transitions associated with its functional properties, i.e., binding to lipid membranes, pore forming activity on lipid vesicles, and hemolysis. At 25 degrees C and between pH 6 and 9, ostreolysin adopted a monomeric and thermodynamically stable nativelike conformation, characterized by rigid tertiary structure and predominantly beta-sheet secondary structure. Between pH 2 and 3, the protein underwent an irreversible transition to a partially unfolded, molten globule-like state which bound ANS, and exhibited disrupted tertiary structure and enhanced non-native alpha-helical structure. Functional studies showed that, unlike colicins and some other bacterial pore-forming toxins, the acid-induced molten globule-like state of ostreolysin is not relevant for lipid binding and pore formation. Instead, the compact native state was necessary for binding to cholesterol/sphingomyelin multilamellar vesicles, optimally in the pH range from 6 to 7, and for pore formation and hemolysis, maximally between pH 7 and 8.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Veèna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Sabina
authors.initials: S
authors.lastname: Berne
authors.forename: Kristina
authors.initials: K
authors.lastname: Sepcić
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.forename: Tom
authors.initials: T
authors.lastname: Turk
authors.forename: Peter
authors.initials: P
authors.lastname: Macek
authors.forename: Natasa
authors.initials: N
authors.lastname: Poklar Ulrih
mesh_terms: D000595:Amino Acid Sequence; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D006461:Hemolysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D020076:Pleurotus / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D017510:Protein Folding; D013109:Sphingomyelins / Q000737:chemistry / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D006460:Hemolysin Proteins; D008567:Membranes, Artificial; D013109:Sphingomyelins; C461280:ostreolysin; D002784:Cholesterol