Slow assembly and disassembly of lambda Cro repressor dimers.

Dimers of Cro are required to recognize operator DNA and repress transcription, but dimerization is weak compared to DNA binding. Fluorophore-conjugated, single-cysteine variants of Cro have been used to investigate the equilibria and kinetics of dimer assembly. Equilibrium distributions of mixed dimers, monitored by fluorescence resonance energy transfer (FRET), confirm that labeled variants have equilibrium dimer dissociation constants in the micromolar concentration range. Subunit exchange experiments yield first order rate constants for dimer dissociation that range from 0.02 s(-1) to 0.04 s(-1). Association rate constants calculated from the ratios of dissociation equilibrium and rate constants range from 0.7x10(4) M(-1) s(-1) to 3x10(4) M(-1) s(-1), depending on the site of the fluorescent label. At nanomolar concentrations of subunits, assembly can be driven by addition of DNA. The bimolecular association rate constants measured under these conditions are not dramatically enhanced, ranging from 7x10(4) M(-1) s(-1) to 9x10(4) M(-1) s(-1). The association rate is second order in protein but independent of DNA concentration between 10 nM and 200 nM. The association of subunits under native conditions is more than four orders of magnitude slower than the fast assembly phase measured previously in refolding experiments, and is unaffected by peptidyl-prolyl isomerases. Stabilization of the folded structure of the protein by residue substitution in Cro F58W or reduced temperature increases the ratio of dimers to monomers and decreases the rate of subunit exchange. These data suggest that native monomers have compact structures with substantial barriers to unfolding and that unfolded or partially folded monomers are the preferred substrates for dimer assembly. Cro binding in vivo may be under kinetic rather than thermodynamic control. The slow assembly of Cro dimers demonstrated here provides a new perspective on the lysis/lysogeny switch of bacteriophage lambda.

Folding and assembly of lambda Cro repressor dimers are kinetically limited by proline isomerization.

Cro binds to operator sites in lambda DNA as a dimer. Dimerization of this small repressor protein is weak, however, and proline residues in the dimer interface suggest that folding and assembly of active repressors may be complex. Cro and selected variants have been studied by circular dichroism and fluorescence. Fluorescent probes include a unique tryptophan residue in the dimer interface and extrinsic resonance energy transfer probes that monitor dimerization. Both folding and unfolding are characterized by two distinct kinetic phases. Fast processes that are complete within the 5-10 ms dead time of stopped flow experiments account for the majority of the change in the CD signal and abrupt changes in both tryptophan fluorescence and energy transfer. The slow phases show all the hallmarks of proline isomerization. The rates of the slow phases are between 0.005 and 0.02 s(-1), are relatively independent of protein and denaturant concentration, display activation energies of 20 kcal/mol, and are accelerated by the peptidyl-prolyl isomerase SlyD. Although CD measurements indicate that more than 70% of the secondary structure is regained in the refolding burst phase, intermolecular fluorescence resonance energy transfer experiments indicate that less than 25% of these subunits are assembled into dimers. Full folding and dimerization requires isomerization of the non-native prolyl isomers over hundreds of seconds.