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. 1996 May;5(5):802–813. doi: 10.1002/pro.5560050502

Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.

F Zappacosta 1, A Pessi 1, E Bianchi 1, S Venturini 1, M Sollazzo 1, A Tramontano 1, G Marino 1, P Pucci 1
PMCID: PMC2143402  PMID: 8732752

Abstract

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.

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Selected References

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