PMID:12860133
| Citation |
Wu, Y and Matthews, CR (2003) Proline replacements and the simplification of the complex, parallel channel folding mechanism for the alpha subunit of Trp synthase, a TIM barrel protein. J. Mol. Biol. 330:1131-44 |
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| Abstract |
The kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli involves four parallel channels whose inter-conversions are controlled by three cis/trans prolyl isomerization reactions (tau(1), tau(2) and tau(3)). A previous mutational analysis of all 19 proline positions, including the unique cis Asp27-Pro28 peptide bond, revealed that the G(3)P28G, P78A or P96A mutations selectively eliminated the fast, tau(1) (ten seconds), folding phase, while the P217M and P261A mutations eliminated the medium, tau(2) (40 seconds) and the slow, tau(3) ( approximately 300 seconds) folding phases, respectively. To further elucidate the role of these proline residues and to simplify the folding mechanism, a series of double and triple mutants were constructed at these critical positions, and comprehensive kinetic and thermodynamic experiments were performed. Although it was not possible to construct a stable system that was free of proline isomerization constraints, a double mutant variant, G(3)P28G/P217M, in which the refolding of more than 90% of the unfolded protein is not limited by proline isomerization reactions was identified. Further, long-range interactions between several of these residues appear to be a crucial part of the cooperative network of structure that stabilizes the TIM barrel motif for alphaTS. |
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| Keywords |
Circular Dichroism; Cyclophilins/pharmacology; DNA Mutational Analysis; Escherichia coli/enzymology; Escherichia coli/metabolism; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Oligonucleotides/chemistry; Proline/chemistry; Protein Binding; Protein Conformation; Protein Folding; Protein Structure, Tertiary; Thermodynamics; Time Factors; Tryptophan Synthase/chemistry; Ultraviolet Rays; Urea/pharmacology |
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