Rescuing a destabilized protein fold through backbone cyclization

Julio A. Camarero, David Fushman, Satoshi Sato, Izabela Giriat, David Cowburn, Daniel P. Raleigh, Tom W. Muir

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

We describe the physicochemical characterization of various circular and linear forms of the ∼60 residue N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein. Structural, dynamic, thermodynamic, kinetic and biochemical studies reveal that backbone circularization does not prevent the adoption of the natural folded structure in any of the circular proteins. Both the folding and unfolding rate of the protein increased slightly upon circularization. Circularization did not lead to a significant thermodynamic stabilization of the full-length protein, suggesting that destabilizing enthalpic effects (e.g. strain) negate the expected favorable entropic contribution to overall stability. In contrast, we find circularization results in a dramatic stabilization of a truncated version of the SH3 domain lacking a key glutamate residue. The ability to rescue the destabilized mutant indicates that circularization may be a useful tool in protein engineering programs geared towards generating minimized proteins.

Original languageEnglish (US)
Pages (from-to)1045-1062
Number of pages18
JournalJournal of Molecular Biology
Volume308
Issue number5
DOIs
StatePublished - May 18 2001
Externally publishedYes

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Cyclization
src Homology Domains
Thermodynamics
Proto-Oncogene Proteins c-crk
Protein Engineering
Protein Unfolding
Proteins
Glutamic Acid

Keywords

  • Circular protein
  • Ligation
  • SH3 domain

ASJC Scopus subject areas

  • Virology

Cite this

Camarero, J. A., Fushman, D., Sato, S., Giriat, I., Cowburn, D., Raleigh, D. P., & Muir, T. W. (2001). Rescuing a destabilized protein fold through backbone cyclization. Journal of Molecular Biology, 308(5), 1045-1062. https://doi.org/10.1006/jmbi.2001.4631

Rescuing a destabilized protein fold through backbone cyclization. / Camarero, Julio A.; Fushman, David; Sato, Satoshi; Giriat, Izabela; Cowburn, David; Raleigh, Daniel P.; Muir, Tom W.

In: Journal of Molecular Biology, Vol. 308, No. 5, 18.05.2001, p. 1045-1062.

Research output: Contribution to journalArticle

Camarero, JA, Fushman, D, Sato, S, Giriat, I, Cowburn, D, Raleigh, DP & Muir, TW 2001, 'Rescuing a destabilized protein fold through backbone cyclization', Journal of Molecular Biology, vol. 308, no. 5, pp. 1045-1062. https://doi.org/10.1006/jmbi.2001.4631
Camarero, Julio A. ; Fushman, David ; Sato, Satoshi ; Giriat, Izabela ; Cowburn, David ; Raleigh, Daniel P. ; Muir, Tom W. / Rescuing a destabilized protein fold through backbone cyclization. In: Journal of Molecular Biology. 2001 ; Vol. 308, No. 5. pp. 1045-1062.
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