Role of the N-terminal region and of beta-sheet residue Thr29 on the activity of the omega2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035.
The dimeric regulatory protein wild-type omega (wt omega2) binds to arrays of 7-bp sequences (heptads) present in the operator DNA region of copy control and partition functions of plasmid pSM19035. Each omega2 protein probably binds with an antiparallel beta-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 omega2T29A both in vivo and in vitro, and reduces the thermodynamic stability deltaG(o)u, but does not change the conformation. Likewise, the binding affinity to DNA is reduced and the association of the two monomeric subunits of the omega2T29A dimer is weakened, as manifested by an increase in the dissociation constant from 3.2 microM for wt omega2 to 6.3 microM for omega2T29A. Denatured dimers are formed upon thermal unfolding of wt omega2 and omega2T29A at ca. 45 microM (D(n)<-->D(u)). Removal of 8 (omega2deltaN8), or even 18 (omega2deltaN18) N-terminal amino acids has no obvious effect either on the core structure or on the activity in comparison to wt omega2. The stability of variants omega2deltaN8 and omega2deltaN18 is similar to that of wt omega2, and their binding to operator DNA is not impaired.
Welfle
Karin
K
Max-Delbrück-Centrum für Molekulare Medizin Berlin-Buch, Robert-Rössle-Str. 10, D-13092 Berlin, Germany.
Pratto
Florencia
F
Misselwitz
Rolf
R
Behlke
Joachim
J
Alonso
Juan C
JC
Welfle
Heinz
H
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009875:Operator Regions, Genetic; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013297:Streptococcus pyogenes / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D013912:Threonine; D014508:Urea