FUNCTION OF THE GREEK KEY CONNECTION ANALYZED USING CIRCULAR PERMUTANTS OF SUPEROXIDE-DISMUTASE

Human Cu,Zn superoxide dismutase (SOD) is a single domain all beta-she et protein with its eight beta-strands arranged as a Greek key beta-ba rrel or immunoglobulin fold. Three circularly permuted variants of SOD were made by joining the native amino- and carboxy-termini, and intro ducing new termini at sites originally within connections between beta -strands. The locations of the new termini were chosen to interrupt be ta-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 ra tes for the purified proteins were similar to those of the native enzy me, indicating that the permutants are correctly folded, Interrupting the covalent cross-bracing provided by the Greek key connection reduce d the stability of the protein by similar to 1.0 kcal/mol, indicating only a slight contribution to conformational stability. The experiment s test and eliminate two hypotheses for folding pathways for Greek key beta-barrels that require N-terminal beta-hairpins or covalent attach ment across the short Greek key connection.