Function of the Greek key connection analysed using circular permutants of superoxide dismutase
| Autor: | John A. Tainer, Maurice Boissinot, Elizabeth D. Getzoff, Diane E. Cabelli, S. Karnas, James R. Lepock, Robert A. Hallewell |
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| Rok vydání: | 1997 |
| Předmět: |
Models
Molecular Protein Folding Protein Conformation Molecular Sequence Data Immunoglobulin domain medicine.disease_cause Protein Structure Secondary General Biochemistry Genetics and Molecular Biology Superoxide dismutase Enzyme Stability medicine Humans Amino Acid Sequence Molecular Biology Escherichia coli chemistry.chemical_classification Calorimetry Differential Scanning General Immunology and Microbiology biology Superoxide Dismutase General Neuroscience Periplasmic space Folding (chemistry) Kinetics Enzyme Biochemistry chemistry Covalent bond Mutagenesis Site-Directed biology.protein Function (biology) Research Article |
| Zdroj: | The EMBO Journal. 16:2171-2178 |
| ISSN: | 0261-4189 |
| DOI: | 10.1093/emboj/16.9.2171 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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