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.
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 x 10(8) M-1 s-1 in 1 M urea at 15 degreesC. 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 domain-not 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.
Gloss
L M
LM
Department of Chemistry, Center for Biomolecular Structure and Function, Pennsylvania State University, University Park 16802, USA.
Matthews
C R
CR
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004058:Diffusion; D019281:Dimerization; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins