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

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Abstract
Human Cu,Zn superoxide dismutase (SOD) is a single domain all β‐sheet protein with its eight β‐strands arranged as a Greek key β‐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 β‐strands. The locations of the new termini were chosen to interrupt β‐turns between the two N‐terminal β‐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 ∼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 β‐barrels that require N‐terminal β‐hairpins or covalent attachment across the short Greek key connection.