title: Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.
abstract: A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a beta-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.
authors.affiliation: Servizio di Spettrometria di Massa del CNR, Napoli, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Zappacosta
authors.forename: A
authors.initials: A
authors.lastname: Pessi
authors.forename: E
authors.initials: E
authors.lastname: Bianchi
authors.forename: S
authors.initials: S
authors.lastname: Venturini
authors.forename: M
authors.initials: M
authors.lastname: Sollazzo
authors.forename: A
authors.initials: A
authors.lastname: Tramontano
authors.forename: G
authors.initials: G
authors.lastname: Marino
authors.forename: P
authors.initials: P
authors.lastname: Pucci
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000378:metabolism*; D007136:Immunoglobulins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007136:Immunoglobulins; C081296:Minibody (WT); D010450:Endopeptidases