Mechanism of Folding of the Dimeric Core Domain of Escherichia coli Trp Repressor: A Nearly Diffusion-Limited Reaction Leads to the Formation of an On-Pathway Dimeric Intermediate

Abstract

A polypeptide corresponding to the core/dimerization domain of E. coli Trp repressor (TR), [2−66]₂ TR, was constructed by insertion of a pair of stop codons into the trpR gene. The kinetic properties of the urea-induced folding of this core fragment were examined by intrinsic tryptophan fluorescence (FL) and circular dichroism (CD) spectroscopy. The kinetic response of wild-type TR (WT TR) is very complicated and has been interpreted to involve three parallel channels with multiple folding and isomerization reactions (Mann et al. (1995) Biochemistry 34, 14573−14580). The refolding of [2−66]₂ TR can be described by a much simpler mechanism, involving an association reaction followed by a urea-dependent first-order folding reaction. The second-order rate constant for the association reaction approaches that of the diffusion limit, 3 × 10⁸ M^-1 s^-1 in 1 M urea at 15 °C. Double-jump experiments demonstrate that ≥93% of the unfolded monomers proceed to the native dimer via the dimeric intermediate; several lines of evidence demonstrate that this dimeric species is an on-pathway intermediate. The subsequent first-order folding reaction of the dimeric intermediate to the native species involves development of additional secondary structure and tertiary structure. The kinetic folding mechanism of [2−66]₂ TR suggests that: (1) the complexity of the folding kinetics of full-length WT TR arises from alternative interactions of the DNA reading heads with the dimerization core domainnot from the intertwined nature of the dimerization interface; (2) residues 2−66 contain all of the sequence information necessary to direct the near-diffusion-limited association reaction in a TR folding reaction; and (3) the formation of secondary and tertiary structure is concurrent with or precedes dimerization, and further development certainly follows the formation of quaternary structure.