Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase from E. coli: fragment complementation and circular permutation reveal stable, alternatively folded forms.

摘要: The effects of chain cleavage and circular permutation on the structure, stability, activity dihydrofolate reductase (DHFR) from Escherichia coli were investigated by various spectroscopic biochemical methods. Cleavage backbone after position 86 resulted in two fragments, {1–86} {87–159}, each which are poorly structured enzymatically inactive. When combined a 1 : molar ratio, however, fragments formed high-affinity (Ka = 2.6 × 107 M−1) complex that displays weakly cooperative urea-induced unfolding transition at micromolar concentrations. retention about 15% enzymatic full-length DHFR is surprising, considering secondary structure substantially reduced its wild-type counterpart. In contrast, circularly permuted form with N-terminus has similar overall stability to DHFR, 50% activity, substantial altered side-chain packing adenosine binding domain, unfolds via an equilibrium intermediate not observed protein. After addition ligand or tight-binding inhibitor methotrexate, both fragment permutant adopt more native-like tertiary structures. These results show changes connectivity can produce alternatively folded forms highlight importance protein-ligand interactions stabilizing active site architecture DHFR.