SIGNIFICANT HYDROGEN-EXCHANGE PROTECTION IN GROEL-BOUND DHFR IS MAINTAINED DURING ITERATIVE ROUNDS OF SUBSTRATE CYCLING

An unresolved key issue in the mechanism of protein folding assisted b y the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. He re we describe the conformational properties of human dihydrofolate re ductase (DHFR) bound to GroEL at different stages of its ATP-driven fo lding reaction, determined by hydrogen exchange labeling and electrosp ray ionization mass spectrometry. Considerable protection involving ab out 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a sm all region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The result s indicate that the extent of protection of the substrate protein rebo und to GroEL is indistinguishable from that of the initial bound state . Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate prot ein during iterative cycling, rather than by complete unfolding of thi s protein substrate on the chaperonin surface.