PMID:9153401
| Citation |
Gloss, LM and Matthews, CR (1997) Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor. Biochemistry 36:5612-23 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1021/bi970056e |
| Keywords |
Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Circular Dichroism; Dimerization; Escherichia coli/chemistry; Escherichia coli/genetics; Escherichia coli/metabolism; Molecular Weight; Peptide Fragments/chemistry; Protein Denaturation; Protein Structure, Tertiary; Repressor Proteins/chemistry; Repressor Proteins/genetics; Repressor Proteins/metabolism; Spectrometry, Fluorescence; Thermodynamics; Tryptophan/chemistry; Urea/chemistry; Urea/metabolism |
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