Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor.
The urea-induced equilibrium unfolding of the Escherichia coli Trp repressor (TR) is a two-state process, involving the native dimeric and unfolded monomeric species. Kinetic studies, however, reveal the presence of transient intermediates that appear only during the folding of the 107-residue protein [Gittelman, M. G., & Matthews, C. R. (1990) Biochemistry 29, 7011-7020]. In order to gain insight into the complex kinetic folding mechanism, the sequence of TR was reduced to the amino-terminal 66 residues, corresponding to the dimerization domain. Two polypeptides, 2-66 and NHis-7-66, were shown to be dimeric at 25 degrees C by size exclusion chromatography and to retain native-like spectroscopic features as evidenced by near- and far-UV circular dichroism and fluorescence spectroscopy. The equilibrium properties of the urea-induced folding of these core fragments were examined by intrinsic tryptophan fluorescence and circular dichroism and found to be well described by a two-state model. At 25 degrees C, the stabilities of both fragments are 14 kcal mol(-1), as compared to the 24 kcal mol(-1) observed for full-length TR. In contrast, the thermal denaturation of [2-66]2 and full-length TR are three-state processes; the midpoint of the transition monitored by absorbance at 292 nm precedes that monitored by circular dichroism at 222 nm. Global analysis of the thermal data as a function of monomer concentration suggests that both the full-length and [2-66]2 TR variants unfold via a dimeric intermediate. Taken together, these results demonstrate that the [2-66]2 fragment constitutes a well-structured, independently folding subdomain of TR that may be useful in elucidating the properties of the transient intermediates observed in the folding of the full-length protein. The dimeric intermediate observed in the thermal denaturation of [2-66]2 suggests that it may be possible to further reduce the core sequence while maintaining the ability to dimerize.
Gloss
L M
LM
Department of Chemistry and Center for Biomolecular Structure and Function, Pennsylvania State University, University Park 16802, USA.
Matthews
C R
CR
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D010446:Peptide Fragments; D012097:Repressor Proteins; D014364:Tryptophan; D014508:Urea; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins