OLIGOMERIZATION DOMAIN-DIRECTED REASSEMBLY OF ACTIVE DIHYDROFOLATE-REDUCTASE FROM RATIONALLY DESIGNED FRAGMENTS

Reassembly of enzymes from peptide fragments has been used as a strate gy for understanding the evolution, folding, and role of individual su bdomains in catalysis and regulation of activity. We demonstrate an ol igomerization-assisted enzyme reassembly strategy whereby fragments ar e covalently linked to independently folding and interacting domains w hose interactions serve to promote efficient refolding and complementa tion of fragments, forming active enzyme. We show that active murine d ihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from compleme ntary N- and C-terminal fragments when fused to homodimerizing GCN4 le ucine zipper-forming sequences as well as heterodimerizing protein par tners. Reassembly is detected by an in vivo selection assay in Escheri chia coil and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic p arameters for the reassembled mutant (Phe-31 --> Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein disse ction and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the str ategy suggests a simple in vivo enzyme-based detection system for prot ein-protein interactions, which we illustrate with two examples: ras-G TPase and raf-ras-binding domain and FK506-binding protein-rapamycin c omplexed with the target of rapamycin TOR2.