Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of minibody

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Abstract
A strategy that combines limited proteolysis experiments and mass spectrometrie 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 β‐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.