Function of the Greek key connection analysed using circular permutants of superoxide dismutase

The EMBO Journal
M BoissinotR A Hallewell

Abstract

Human Cu,Zn superoxide dismutase (SOD) is a single domain all beta-sheet protein with its eight beta-strands arranged as a Greek key beta-barrel or immunoglobulin fold. Three circularly permuted variants of SOD were made by joining the native amino- and carboxy-termini, and introducing new termini at sites originally within connections between beta-strands. The locations of the new termini were chosen to interrupt beta-turns between the two N-terminal beta-hairpins and the short cross-barrel Greek key connection. Expression levels in the Escherichia coli periplasm were indistinguishable from that of native SOD. Reaction rates for the purified proteins were similar to those of the native enzyme, indicating that the permutants are correctly folded. Interrupting the covalent cross-bracing provided by the Greek key connection reduced the stability of the protein by approximately 1.0 kcal/mol, indicating only a slight contribution to conformational stability. The experiments test and eliminate two hypotheses for folding pathways for Greek key beta-barrels that require N-terminal beta-hairpins or covalent attachment across the short Greek key connection.

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Citations

Sep 10, 2003·Journal of Molecular Biology·Michael DiDonatoJohn A Tainer
Aug 28, 2003·Bioorganic Chemistry·James B GarrettFrank M Raushel
Feb 4, 2009·Proceedings of the National Academy of Sciences of the United States of America·Tomoyuki AwanoJoan R Coates
Nov 15, 2003·Proceedings of the National Academy of Sciences of the United States of America·Steen V PetersenJan J Enghild
Aug 25, 2010·The Veterinary Clinics of North America. Small Animal Practice·Joan R Coates, Fred A Wininger
Nov 17, 2009·Biochimica Et Biophysica Acta·J J P PerryJ A Tainer
Jul 4, 2006·Biochimica Et Biophysica Acta·Rishi Rakhit, Avijit Chakrabartty
Nov 5, 2016·Protein Science : a Publication of the Protein Society·Yang YuYi Lu

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