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Combinations of the alpha-helix-turn-alpha-helix motif of TetR with respective residues from LacI or 434Cro: DNA recognition, inducer binding, and urea-dependent denaturation.
Biochemistry. 1997 May 06; 36(18):5311-22.B

Abstract

We constructed 10 different variants of TetR by substituting all or some of the residues in the alpha-helix-turn-alpha-helix (HTH) operator binding motif with the respective amino acids from LacI or 434Cro. The variants were soluble, negative transdominant over tetR in vivo, and as active as wild-type TetR in tetracycline binding in vitro. The urea-induced denaturation of the 10 variants occurs in single reversible transitions, which are centered around 4.3 M urea. Denaturation is concentration-dependent, supporting a simple two-state mechanism in which the folded dimeric protein is in equilibrium with unfolded monomers. An analysis according to the two-state model yields a Gibbs free energy of stabilization (at 0 M urea, 25 degrees C) of about 75 kJ/mol, typical for dimeric proteins of this size. Even a deletion of 24 residues from the reading head decreased the stability by only 2.7 kJ/mol. These results suggest that the DNA reading head of Tet repressor is a thermodynamically independent domain and that the thermodynamic stability of the Tet repressor dimer is determined by the association of the dimerization domains of the individual monomers. Variants containing replacements in the first alpha-helix of HTH did not show any DNA binding activity whatsoever. We attribute this to the alteration of the two N-terminal residues in this alpha-helix. TetR variants were active in nonspecific DNA binding, when either all or only the solvent-exposed residues in the recognition alpha-helix of HTH were exchanged to the respective LacI sequence. Replacement of the same residues by the respective amino acids from 434Cro yielded hybrid proteins that specifically recognize tetO in vitro. Taken together, these results establish that the similarity of operator recognition between 434Cro and TetR is greater than between TetR and LacI and confirm that prediction of the recognized DNA sequence is not obvious from the sequence of the respective HTH or recognition alpha-helix.

Authors+Show Affiliations

Lehrstuhl für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9154913

Citation

Backes, H, et al. "Combinations of the Alpha-helix-turn-alpha-helix Motif of TetR With Respective Residues From LacI or 434Cro: DNA Recognition, Inducer Binding, and Urea-dependent Denaturation." Biochemistry, vol. 36, no. 18, 1997, pp. 5311-22.
Backes H, Berens C, Helbl V, et al. Combinations of the alpha-helix-turn-alpha-helix motif of TetR with respective residues from LacI or 434Cro: DNA recognition, inducer binding, and urea-dependent denaturation. Biochemistry. 1997;36(18):5311-22.
Backes, H., Berens, C., Helbl, V., Walter, S., Schmid, F. X., & Hillen, W. (1997). Combinations of the alpha-helix-turn-alpha-helix motif of TetR with respective residues from LacI or 434Cro: DNA recognition, inducer binding, and urea-dependent denaturation. Biochemistry, 36(18), 5311-22.
Backes H, et al. Combinations of the Alpha-helix-turn-alpha-helix Motif of TetR With Respective Residues From LacI or 434Cro: DNA Recognition, Inducer Binding, and Urea-dependent Denaturation. Biochemistry. 1997 May 6;36(18):5311-22. PubMed PMID: 9154913.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Combinations of the alpha-helix-turn-alpha-helix motif of TetR with respective residues from LacI or 434Cro: DNA recognition, inducer binding, and urea-dependent denaturation. AU - Backes,H, AU - Berens,C, AU - Helbl,V, AU - Walter,S, AU - Schmid,F X, AU - Hillen,W, PY - 1997/5/6/pubmed PY - 1997/5/6/medline PY - 1997/5/6/entrez SP - 5311 EP - 22 JF - Biochemistry JO - Biochemistry VL - 36 IS - 18 N2 - We constructed 10 different variants of TetR by substituting all or some of the residues in the alpha-helix-turn-alpha-helix (HTH) operator binding motif with the respective amino acids from LacI or 434Cro. The variants were soluble, negative transdominant over tetR in vivo, and as active as wild-type TetR in tetracycline binding in vitro. The urea-induced denaturation of the 10 variants occurs in single reversible transitions, which are centered around 4.3 M urea. Denaturation is concentration-dependent, supporting a simple two-state mechanism in which the folded dimeric protein is in equilibrium with unfolded monomers. An analysis according to the two-state model yields a Gibbs free energy of stabilization (at 0 M urea, 25 degrees C) of about 75 kJ/mol, typical for dimeric proteins of this size. Even a deletion of 24 residues from the reading head decreased the stability by only 2.7 kJ/mol. These results suggest that the DNA reading head of Tet repressor is a thermodynamically independent domain and that the thermodynamic stability of the Tet repressor dimer is determined by the association of the dimerization domains of the individual monomers. Variants containing replacements in the first alpha-helix of HTH did not show any DNA binding activity whatsoever. We attribute this to the alteration of the two N-terminal residues in this alpha-helix. TetR variants were active in nonspecific DNA binding, when either all or only the solvent-exposed residues in the recognition alpha-helix of HTH were exchanged to the respective LacI sequence. Replacement of the same residues by the respective amino acids from 434Cro yielded hybrid proteins that specifically recognize tetO in vitro. Taken together, these results establish that the similarity of operator recognition between 434Cro and TetR is greater than between TetR and LacI and confirm that prediction of the recognized DNA sequence is not obvious from the sequence of the respective HTH or recognition alpha-helix. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9154913/Combinations_of_the_alpha_helix_turn_alpha_helix_motif_of_TetR_with_respective_residues_from_LacI_or_434Cro:_DNA_recognition_inducer_binding_and_urea_dependent_denaturation_ L2 - https://doi.org/10.1021/bi961527k DB - PRIME DP - Unbound Medicine ER -