Transcriptional repressor CopR: the structured acidic C terminus is important for protein stability11Edited by J. Karn

Kuhn, Kornelia; Steinmetzer, Katrin; Brantl, Sabine

doi:10.1006/jmbi.2000.3929

Cited by 13 publications

(17 citation statements)

References 30 publications

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“…Plasmids pCOPVT, pCOPVT∆7 and the plasmids containing individual E to K substitutions were constructed using a single-step PCR on plasmid pCOP7 as template with primer 486-34 (Kuhn et al, 2000) and one of the mutagenic primers described in Table 1. The resulting 500 bp PCR fragment was digested with BamHI and inserted into the single BamHI site of plasmid pPR7 (Brantl & Behnke, 1992).…”

Section: Construction Of Escherichia Coli/b Subtilis Shuttle Vectorsmentioning

confidence: 99%

“…These were performed as described previously (Kuhn et al, 2000). In all cases, 100 µl polyclonal antiserum raised against His ' -CopR purified from E. coli were used for the precipitation of mutated CopR proteins from the lysates.…”

Section: Construction Of E Coli Vectors For Overexpression Of Mutatedmentioning

confidence: 99%

“…To investigate the importance of the C-terminal 7 aa residues located downstream of the β-strand forming sequence, pCOPVT∆7 was constructed. To analyse the importance of the previously found β-strand forming sequence (Kuhn et al, 2000). QVTLELEME between aa 76 and 84 of CopR, plasmid pCOPVT was constructed encoding a CopR protein where this sequence was replaced by QVTVTVTVT, a sequence which Brack & Caille (1978) had shown that to be present in the β conformation in solution.…”

Section: Design and Analysis Of C-terminally Substituted Copr Mutantsmentioning

confidence: 99%

“…Previously, we showed that CopR binds asymmetrically as a preformed dimer at two consecutive major grooves of the DNA (Steinmetzer & Brantl, 1997 ;Steinmetzer et al, 1998) and identified amino acids involved in DNA binding and dimerization (Steinmetzer et al, 2000a, b). The last 29 aa of CopR are essential for neither DNA binding nor dimerization, but are essential for protein stability (Kuhn et al, 2000). The half-life of CopR amounts to 42p5 min.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, we focused our interest on the last 20 aa. Circular dichroism (CD)-difference spectra revealed that this sequence is not unstructured, but part of it, most probably between aa 76 and 84 (QVTLELEME) apparently forms a β-strand with alternating hydrophilic and hydrophobic residues (Kuhn et al, 2000). This β-strand forming sequence is also present in the related repressors CopF (Swinfield et al, 1990) and CopS (Ceglowski & Alonso, 1994), albeit as QVTLDLEME.…”

Section: Introductionmentioning

confidence: 99%

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Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

2001

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Replication of the streptococcal plasmid pIP501 is regulated by two components, CopR and the antisense RNA, RNAIII. CopR represses transcription of the essential repR mRNA about 10-to 20-fold and, additionally, prevents convergent transcription of sense and antisense RNAs. It has been demonstrated that CopR binds as a preformed dimer. DNA binding and dimerization constants were determined and amino acids were identified that are involved in DNA binding and dimerization. It was demonstrated that the Cterminal 20 aa of CopR are not involved in either activity, but play an important role for CopR stability. Furthermore, it was found that the C terminus of CopR is structured containing a β-strand structure, most probably between the alternating hydrophilic and hydrophobic amino acids 76 and 84 (QVTLELEME). In this study stability motifs within the C terminus of CopR were dissected. Both the cognate and a heterologous (QVTVTVTVT) β-strand structure between amino acids 76 and 84 within the C terminus stabilized CopR (CopR derivative CopVT). In contrast, substitution by a predicted α-helix (QVTLKLKMK) or a predicted unstructured sequence (QVTPEPEPE) caused severe and moderate destabilization, respectively. E80 seemed to be the only important C-terminal glutamic acid residue. Deletion of seven C-terminal amino acids from either wild-type CopR or CopVT reduced the half-life to " 50 % indicating that this C-terminal sequence is a second stability motif.

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Section: Construction Of Escherichia Coli/b Subtilis Shuttle Vectorsmentioning

confidence: 99%

Section: Construction Of E Coli Vectors For Overexpression Of Mutatedmentioning

confidence: 99%

Section: Design and Analysis Of C-terminally Substituted Copr Mutantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

2001

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Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix–helix interactions

et al. 2003

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Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1-75), the addition of linker residues 76 to 80 (MKEQD) raised the Tm by 9 degrees C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., [Biochemistry 2002;41:15-20]), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1-74 to PCaM1-80). For the first 4, PCaM1-74 through PCaM1-77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5 degrees C in Tm and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1-77 through PCaM1-80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of 15N-PCaM1-74 and 15N-PCaM1-80 and analysis of high-resolution structural models suggest these residues are latched to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains.

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Antisense-RNA mediated control of plasmid replication – pIP501 revisited

Brantl

2015

Plasmid

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Transcriptional repressor CopR: the structured acidic C terminus is important for protein stability11Edited by J. Karn

Cited by 13 publications

References 30 publications

Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix–helix interactions

Antisense-RNA mediated control of plasmid replication – pIP501 revisited

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