PROBING THE TERTIARY STRUCTURE OF PROTEINS BY LIMITED PROTEOLYSIS ANDMASS-SPECTROMETRY - THE CASE OF MINIBODY

A strategy that combines limited proteolysis experiments and mass spec trometric analysis of the fragments generated has been developed to pr obe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of th e 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 th e hypervariable H1 and H2 regions. The low solubility of the protein p revented a detailed characterization by NMR and/or X-ray. Different pr oteases were used under strictly controlled conditions and the cleavag e sites were mapped onto the anticipated Minibody model, leading to th e identification of the most exposed regions, A single-residue mutant was constructed and characterized, following the same procedure, showi ng a slightly higher correspondence with the predicted model. This str ategy can be used to effectively supplement NMR and X-ray investigatio ns of protein tertiary structure, where these procedures cannot provid e definitive data, or to verify and refine protein models.