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Karin Welfle, Florencia Pratto, Rolf Misselwitz, Joachim Behlke, Juan C. Alonso, Heinz Welfle

Role of the N-terminal region and of ?-sheet residue Thr29 on the activity of the ?₂ global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035

Keywords: MetJ/Arc superfamily, protein-DNA interaction, protein folding, Thermodynamics, transcriptional repressor

The dimeric regulatory protein wild-type ? (wt ?₂) binds to arrays of 7-bp sequences (heptads) present in the operator DNA region of copy control and partition functions of plasmid pSM19035. Each ?₂ protein probably binds with an antiparallel ?-sheet structure in the major groove of the 7-bp subsite of the operator DNA. Exchange of threonine at position 29 to alanine (T29A) drastically affects the activity of variant protein ?₂T29A both in vivo and in vitro, and reduces the thermodynamic stability ?G_u⁰, but does not change the conformation. Likewise, the binding affinity to DNA is reduced and the association of the two monomeric subunits of the ?₂T29A dimer is weakened, as manifested by an increase in the dissociation constant from 3.2 ?M for wt ?₂ to 6.3 ?M for ?₂T29A. Denatured dimers are formed upon thermal unfolding of wt ?₂ and ?₂T29A at ca. 45 ?M (D_n?D_u). Removal of 8 (?₂?N8), or even 18 (?₂?N18) N-terminal amino acids has no obvious effect either on the core structure or on the activity in comparison to wt ?₂. The stability of variants ?₂?N8 and ?₂?N18 is similar to that of wt ?₂, and their binding to operator DNA is not impaired.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 386, 09/2005
Pages: 881 - 894

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