Surface topology of Minibody by selective chemical modifications and mass spectrometry.
The surface topology of the Minibody, a small de novo-designed beta-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study indicate that, while overall the Minibody model is correct, the beta-sheet formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini S, Sollazzo M, Tramontano A. Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5:802-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.
Zappacosta
F
F
Centro Internazionale, CNR-Università di Napoli, Italy.
Ingallinella
P
P
Scaloni
A
A
Pessi
A
A
Bianchi
E
E
Sollazzo
M
M
Tramontano
A
A
Marino
G
G
Pucci
P
P
D000107:Acetylation; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001120:Arginine / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007136:Immunoglobulins / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007136:Immunoglobulins; C081296:Minibody (WT); D010446:Peptide Fragments; D011994:Recombinant Proteins; D014443:Tyrosine; D001120:Arginine; D008239:Lysine