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In vivo protein cyclization promoted by a circularly-permuted Synechocystis sp. PCC6803 DnaB mini-intein
Faculty of Science, Medicine and Health - Papers
  • Neal K Williams, Australian National University
  • Pavel Prosselkov, Australian National University
  • Edvards Liepinsh, Karolinska Institutet
  • Inara Line, Latvian University
  • Anatoly Sharipo, Latvian University
  • Dene R Littler, University of New South Wales
  • Paul M.G Curmi, University Of New South Wales
  • Gottfried Otting, Karolinska Institutet
  • Nicholas E Dixon, University of Wollongong
RIS ID
16972
Publication Date
1-1-2002
Publication Details

Williams, N. K., Prosselkov, P., Liepinsh, E., Line, I., Sharipo, A., Littler, D. R., Curmi, P. M.G., Otting, G. & Dixon, N. E. (2002). In vivo protein cyclization promoted by a circularly-permuted Synechocystis sp. PCC6803 DnaB mini-intein. Journal of Biological Chemistry, 277 (10), 7790-7798.

Abstract
A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivocyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures ∼14 °C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2 and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (ΔΔG ≈ 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.
Citation Information
Neal K Williams, Pavel Prosselkov, Edvards Liepinsh, Inara Line, et al.. "In vivo protein cyclization promoted by a circularly-permuted Synechocystis sp. PCC6803 DnaB mini-intein" (2002)
Available at: http://works.bepress.com/nick_dixon/41/