Preceding hydrophobic and β-branched amino acids attenuate splicing by the CnePRP8 intein

Pearl, Esther J.; Bokor, Annika A.M.; Butler, Margaret I.; Poulter, Russell T. M.; Wilbanks, Sigurd M.

doi:10.1016/j.bbapap.2007.05.015

Cited by 17 publications

(11 citation statements)

References 29 publications

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“…Moreover, Cne PRP8 intein was mostly impaired by mutations at the −1 position of the N-extein, when β-branched amino acid residues were incorporated, which is in good agreement with Pho RadA intein and Sce VMA intein (Chong et al 1998b;Oeemig et al 2012;Pearl et al 2007). Pto VMA intein contains a lysine residue, which was shown to be predominant with glycine at position −1 in inteins that have been identified so far (Amitai et al 2009;Perler 2002).…”

Section: Discussionsupporting

confidence: 79%

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A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

Heyde

Lockhauserbäumer

Uetrecht

et al. 2015

Appl Microbiol Biotechnol

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Extein amino acid residues around the splice site junctions affect the functionality of inteins. To identify an optimal sequence context for efficient protein splicing of an intein from the thermoacidophilic archaeon Picrophilus torridus, single extein amino acid residues at the splice site junctions were continuously deleted. The construction of a set of different truncated extein variants showed that this intein tolerates multiple amino acid variations near the excision sites and exhibits full activity when -1 and +1 extein amino acid residues are conserved in an artificial GST-intein-HIS fusion construct. Moreover, splicing of the recombinant intein took place at temperatures between 4 and 42 °C with high efficiency, when produced in Escherichia coli. Therefore, structural model predictions were used to identify optimal insertion sites for the intein to be embedded within a hemicellulase from the psychrophilic bacterium Pseudoalteromonas arctica. The P. torridus intein inserted before amino acid residue Thr75 of the reporter enzyme retained catalytic activity. Moreover, the catalytic activity of the xylan-degrading hydrolase could be easily monitored in routine plate assays and in liquid test measurements at room temperature when produced in recombinant form in E. coli. This tool allows the indirect detection of the intein's catalytic activity to be used in screenings.

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Section: Discussionsupporting

confidence: 79%

“…d Plate assays for the detection of xylanase activity. Cells were grown on LB-medium containing IPTG and overlayed with AZCL-dye coupled arabinoxylan intein insertion site Appleby-Tagoe et al 2011;Ellilä et al 2011;Pearl et al 2007;Shen et al 2012). …”

Section: Discussionmentioning

confidence: 99%

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

Heyde

Lockhauserbäumer

Uetrecht

et al. 2015

Appl Microbiol Biotechnol

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“…There are specific characteristics for intein insertion sites that allow functional splicing [11], [12], [13], [14], [15]. This has been demonstrated by several experiments including one study where different portions of the intein’s native exteins are transferred with the intein [15], and mutagenesis studies that have shown the preference for the local sequence context [16], preference for particular flanking extein residues [15], [17], [18], [19] and the preference for inteins to splice out of some but not all sites in a target protein [11], [12], [13], [14], [15], [20].…”

Section: Introductionmentioning

confidence: 99%

A Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches

et al. 2012

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Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch.

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“…While there is general agreement on the overall pathway leading from an intein precursor to spliced products6, many fundamental questions remain. These include how the intein rearranges a stable peptide bond into a reactive thioester to initiate splicing, and the precise relationship between the intein and the flanking amino- and carboxyl-terminal sequences, called N- and C-exteins, respectively7–9. Experiments to interrogate these issues are stymied by the difficulties of isolating unspliced precursor and by the instability of protein splicing intermediates10.…”

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confidence: 99%

Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications

Callahan

Topilina

Stanger

et al. 2011

Nat Struct Mol Biol

102

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Inteins self-splice from precursor polypeptides to reconstitute functional proteins. Here we describe inteins as redox-responsive switches in bacteria. Regulation was achieved by engineering a disulfide bond between the intein’s catalytic cysteine and the flanking polypeptide. This interaction was validated by an X-ray structure, which includes a transient splice junction. A natural analogue of the designed system was identified in Pyrococcus abysii, suggesting an unprecedented form of adaptive, post-translational regulation.

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Preceding hydrophobic and β-branched amino acids attenuate splicing by the CnePRP8 intein

Cited by 17 publications

References 29 publications

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

A Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches

Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications

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