Isolation and analysis of arc repressor mutants: Evidence for an unusual mechanism of DNA binding

Vershon, Andrew K.; Bowie, James U.; Karplus, Theresa M.; Sauer, Robert T.

doi:10.1002/prot.340010404

Cited by 75 publications

(100 citation statements)

References 28 publications

(3 reference statements)

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“…Two factors, thermodynamic stability and C-terminal sequence, appear to determine the susceptibility of Arc and its variants to intracellular degradation (Vershon et al, 1986;Bowie & Sauer, 1989b;Parsell & Sauer, 1989;Parsell et al, 1990). Protein stability is thought to be impor- tant because many mutations that reduce thermodynamic stability also lead to increased degradation in the cell.…”

Section: Discussionmentioning

confidence: 99%

“…To address this issue, we assayed the activities of the nine mutants described above and an additional seven mutants from the collection of Vershon et al (1986) in the st5 tail background. As described in Materials and methods, an Arc+ phenotype in strain UA2F results in chloramphenicol sensitivity, whereas an Arc-phenotype results in a chloramphenicol resistance.…”

Section: Increased Expression Can Suppress Some But Not All Mutant Dementioning

confidence: 99%

“…Proteolysis-sensitive Arc mutations were recloned from the collection of Vershon et al (1986) into different tail backgrounds. The original mutations were isolated in plasmid pTA200.…”

Section: Strains and Plasmidsmentioning

confidence: 99%

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P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences

1993

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Many mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli, which precludes their expression at levels sufficient for purification and subsequent biochemical characterization. Here we examine the effects of several different C-terminal extension sequences on the expression and activity of a set of Arc mutants. We show that two tail sequences, KNQHE (st5) and H,KNQHE (st1 I), increase the expression levels of most mutants from 10-to 20-fold and, in some cases, result in restoration of biological activity in the cell. A third tail sequence, H H H H H H (st@, was not as effective in increasing mutant expression levels. All three tail sequences are functionally and structurally silent, as judged by their lack of effects on the DNA binding activity and stability of otherwise wild-type Arc. The properties of the st1 1 tail sequence make it an efficient system for the expression and purification of mutant Arc proteins, both because mutant expression levels are increased and because the proteins can be rapidly purified using nickel-chelate affinity chromatography. Arc mutants containing the EA28, RL31, and SA32 mutations were purified in the st1 1 background. The thermodynamic stability of the EA28 mutant (AAG, = -0.4 kcal/mol) is reduced modestly compared to the st1 1 parent, whereas the RL31 mutant (AAG, = -3.0 kcal/mol) and SA32 mutant (AAG, = -3.3 kcal/mol) are substantially less stable.

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Section: Discussionmentioning

confidence: 99%

Section: Increased Expression Can Suppress Some But Not All Mutant Dementioning

confidence: 99%

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P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences

1993

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“…Destabilizing substitutions often result in low or undetectable levels of the mutant protein in the aqueous fraction of cell lysates. Such mutants are either found in insoluble inclusion bodies or subjected to proteolysis in vivo (24,25). It was therefore of interest to examine the solubilities of the various Asp mutants.…”

Section: Correlation Of Solubility Of Asp Mutants With Residuementioning

confidence: 99%

Mutagenesis-based definitions and probes of residue burial in proteins

Bajaj

Chakrabarti²,

Varadarajan³

2005

Proc. Natl. Acad. Sci. U.S.A.

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Every residue of the 101-aa Escherichia coli toxin CcdB was substituted with Ala, Asp, Glu, Lys, and Arg by using site-directed mutagenesis. The activity of each mutant in vivo was characterized as a function of Controller of Cell Division or Death B protein (CcdB) transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, maltose-binding protein and thioredoxin, also were shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose-dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. With continuous reductions in oligonucleotide costs and increasingly efficient site-directed mutagenesis procedures, comprehensive scanning mutagenesis experiments for small proteins͞domains are quite feasible.accessibility ͉ aspartate ͉ scanning mutagenesis ͉ phenotype

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“…Plasmids were introduced by electroporation or M13-mediated transduction (Vershon et al, 1986) into E. coli strain AG1688 [F'128 la& lacZ::TnS/araD139, A(araleu17697, A(lac)X74, galE15, galKl6, rpsL(StrR), hsdR2, mcrA, mcrBI] (Hu et al, 1993) or derivatives bearing reporter genes as lysogens of specialized transducing phages. JH607 and XZ980 are lysogens of A1 120,P, and AXZ970, respectively, in AG1688 .…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Oligomerization properties of GCN4 leucine zipper e and g position mutants

et al. 1997

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Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g-e' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of >65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing A repressor-leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher-order (>dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type I11 mutant formed a variety of higher-order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g-e' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.Keywords: dimerization specificity; leucine zippers; protein structure; recombinant fusion proteins; site-directed mutagenesis a-Helical coiled coils are involved in the assembly of a wide variety of proteins. A subclass of the coiled-coils known as leucine zippers are found as short dimerization motifs in many eukaryotic transcription factors. Leucine zipper sequences are characterized by leucine appearing in every seventh position (4 over four to five heptad repeats (abcdefg), (for a review, see Hurst, 1994). Leucine zippers fold into dimeric, parallel coiled-coils, where each heptad forms two a-helical turns. Because of their small size and simple structure, leucine zippers and other short a-helical coiled coils have been used extensively as a model system to study how amino acid sequences specify structure (e.g., see Hodges et al., 1988;

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Isolation and analysis of arc repressor mutants: Evidence for an unusual mechanism of DNA binding

Cited by 75 publications

References 28 publications

P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences

P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences

Mutagenesis-based definitions and probes of residue burial in proteins

Oligomerization properties of GCN4 leucine zipper e and g position mutants

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