Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.
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.
Zappacosta
F
F
Servizio di Spettrometria di Massa del CNR, Napoli, Italy.
Pessi
A
A
Bianchi
E
E
Venturini
S
S
Sollazzo
M
M
Tramontano
A
A
Marino
G
G
Pucci
P
P
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
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007136:Immunoglobulins; C081296:Minibody (WT); D010450:Endopeptidases