title: Limited proteolysis of cytochrome c in trifluoroethanol.
abstract: Horse heart cytochrome c is cleaved by thermolysin in 50% aqueous TFE (v/v) at neutral pH (25 degrees C, 24 h) at the Gly56-Ile57 peptide bond of the 104-residue chain of the protein. Additional, but anyway minor, fragmentation at the Gly45-Phe46 and Met80-Ile81 peptide bonds is also observed. On the other hand, in buffer only and in the absence of TFE, cytochrome c is digested by thermolysin to numerous small peptides. Considering the broad substrate specificity of the TFE-resistant thermolysin, clearly the conformational state of the protein substrate dictates the observed selective proteolysis. It is proposed that the highly helical secondary structure acquired by cytochrome c when dissolved in aqueous TFE hampers binding and adaptation of the protein substrate at the active site of the protease and that peptide bond fission occurs at flexible chain segments characterized by a low alpha-helix propensity.
authors.affiliation: CRIBI Biotechnology Centre, University of Padua, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Fontana
authors.forename: M
authors.initials: M
authors.lastname: Zambonin
authors.forename: V
authors.initials: V
authors.lastname: De Filippis
authors.forename: M
authors.initials: M
authors.lastname: Bosco
authors.forename: P
authors.initials: P
authors.lastname: Polverino de Laureto
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D008969:Molecular Sequence Data; D009206:Myocardium; D010446:Peptide Fragments / Q000096:biosynthesis / Q000302:isolation & purification; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013820:Thermolysin / Q000378:metabolism*; D014270:Trifluoroethanol
chemical_list: D000596:Amino Acids; D003574:Cytochrome c Group; D010446:Peptide Fragments; D014270:Trifluoroethanol; D013820:Thermolysin