Rough energy landscapes in protein folding: Dimeric E-coliTrp repressor folds through three parallel channels

The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages o f development. Following the formation of a partially folded, monomeric ens emble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non -proline isomerization. reaction or a dimerization reaction, depending on t he final denaturant concentration. Two approaches have been employed to tes t the previously proposed folding mechanism of TR through three parallel ch annels: (1) unfolding double-jump experiments demonstrate that all three fo lding channels lead directly to native dimer; and (2) the differential stab ilization of the transition state for the final step in folding and the nat ive dimer, by the addition of salt, shows that all three channels involve i somerization of a dimeric species. A refined model for the folding of Trp r epressor is presented, in which all three channels involve a rapid dimeriza tion reaction between partially folded monomers followed by the isomerizati on of the dimeric intermediates to yield native dimer. The ensemble of part ially folded monomers can be captured at equilibrium by low pH; one-dimensi onal proton NMR spectra at pH 2.5 demonstrate that monomers exist in two di stinct, slowly interconverting conformations. These data provide a potentia l structural explanation for the three-channel folding mechanism of TR: ran dom association of two different monomeric forms, which are distinguished b y alternative packing modes of the core dimerization domain and the DNA-bin ding, helix-turn-helix, domain. One, perhaps both, of these packing modes c ontains non-native contacts. (C) 2001 Academic Press.