SURFACE-TOPOLOGY OF MINIBODY BY SELECTIVE CHEMICAL MODIFICATIONS AND MASS-SPECTROMETRY

The surface topology of the Minibody, a small de novo-designed beta-pr otein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysi s of the modified fragments. Under appropriate conditions, the suscept ibility of individual residues primarily depends on their surface acce ssibility so that their relative reactivities can be correlated with t heir position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramol ecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study in dicate that, while overall the Minibody model is correct, the beta-she et formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained usi ng a similar approach where mass spectrometry was used to identify Min ibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianc hi E, Venturini S, Sollazzo M, Tramontano A, Ma?ino 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 c hemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural informatio n to complement simulation techniques. This is especially valuable whe n, as in the Minibody case, an NMR and/or X-ray structure cannot be ob tained due to insufficient solubility of the molecule.