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Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor.
Biochemistry. 1997 May 13; 36(19):5612-23.B

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.

Authors+Show Affiliations

Department of Chemistry and Center for Biomolecular Structure and Function, Pennsylvania State University, University Park 16802, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9153401

Citation

Gloss, L M., and C R. Matthews. "Urea and Thermal Equilibrium Denaturation Studies On the Dimerization Domain of Escherichia Coli Trp Repressor." Biochemistry, vol. 36, no. 19, 1997, pp. 5612-23.
Gloss LM, Matthews CR. Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor. Biochemistry. 1997;36(19):5612-23.
Gloss, L. M., & Matthews, C. R. (1997). Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor. Biochemistry, 36(19), 5612-23.
Gloss LM, Matthews CR. Urea and Thermal Equilibrium Denaturation Studies On the Dimerization Domain of Escherichia Coli Trp Repressor. Biochemistry. 1997 May 13;36(19):5612-23. PubMed PMID: 9153401.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor. AU - Gloss,L M, AU - Matthews,C R, PY - 1997/5/13/pubmed PY - 1997/5/13/medline PY - 1997/5/13/entrez SP - 5612 EP - 23 JF - Biochemistry JO - Biochemistry VL - 36 IS - 19 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9153401/Urea_and_thermal_equilibrium_denaturation_studies_on_the_dimerization_domain_of_Escherichia_coli_Trp_repressor_ L2 - https://doi.org/10.1021/bi970056e DB - PRIME DP - Unbound Medicine ER -