Plasmid pIP501 encoded transcriptional repressor CopR: single amino acids involved in dimerization are also important for folding of the monomer.
CopR is one of the two components regulating replication of plasmid pIP501. It binds as a preformed dimer at the DNA major groove thereby repressing transcription of the essential repR-mRNA 10-20-fold. Previously, the DNA-binding motif was identified and the location of the dimeric interface was narrowed down. The C-terminal 29 residues were shown to be required exclusively for CopR stability. Here, we report the characterization of four single amino acid exchange mutants at the dimeric interface. All mutants were inactive in copy number control in vivo. Dimerization constants and DNA-binding constants were determined by analytical ultracentrifugation and EMSA, respectively. Denaturation experiments were performed to estimate the protein stability and to calculate DeltaG(0)(H(2)O). Our data indicate that the four analyzed amino acids are both involved in dimerization and proper folding of the monomer; i.e. they stabilize on the one hand the monomer and on the other hand the dimeric interface.
Steinmetzer
Katrin
K
Institut für Molekularbiologie, Friedrich-Schiller-Universität Jena, Winzerlaer Str. 10, Jena, D-07745, Germany.
Kuhn
Kornelia
K
Behlke
Joachim
J
Golbik
Ralph
R
Brantl
Sabine
S
D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D014508:Urea; D004247:DNA