Protein splicing is a self‐catalyzed reaction by which an intein, an intervening polypeptide, catalyzes its own removal from exteins, the flanking polypeptides, as well as ligation of the exteins. We are studying the inteins that interrupt a cell division control protein in the halophilic archaea Haloquadratum walsbyi. Halophiles like H. walsbyi are able to survive in water with high concentrations of sodium chloride. H. walsbyi has four inteins that interrupt the cdc21 gene, and we have studied the two inteins (Hwa cdc21A and Hwa cdc21D) that lack an intervening homing endonuclease domain. We are studying the splicing activity under various salt concentrations for the inteins both separately and when expressed as part of a single fusion protein. Hwa cdc21A splices optimally at 2.5 M NaCl, while Hwa cdc21D splices optimally at 1.5 M NaCl. We also are using native Trp fluorescence to study the influence of salt concentration on the proper folding of the inteins. Support or Funding Information This work was supported by NSF grant MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award, and NIH Grant 1R15GM132817‐01.
Protein splicing is a self‐catalyzed, post‐translational process in which an intervening polypeptide sequence, or an intein, catalyzes its removal from two flanking polypeptide sequences, or exteins, as well as the ligation of the exteins. We are interested in the splicing capabilities of two inteins that interrupt the cdc21 protein from Haloquadratum walsbyi (Hwa). Hwa is a halophilic archaeon found in high salt environments such as the Red Sea. For this reason, we hypothesize that protein splicing may be salt dependent. We are interested in studying the splicing capabilities of both inteins separately and when expressed as part of the same protein. One intein, Cdc21a, does not splice on overexpression in E. coli, but can be induced to splice in vitro on incubation with 2.5 M sodium chloride at 28°. The other intein, Cdc21d, splices in vivo, and can be induced to splice further with higher concentrations of salt. When expressed in the same precursor fusion protein, the inteins continue to show differential activity conditional on salt concentration, leading to alternative spliced products.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM), the Dreyfus Foundation (KVM), and Diane D. Brink P12 to the Alumni/Parent Summer Research Scholarship Fund. ALThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein splicing is a post-translational process by which an intervening protein, or an intein, catalyzes its own excision from flanking polypeptides, or exteins, coupled to extein ligation. Four inteins interrupt the MCM helicase of the halophile Haloquadratum walsbyi, two of which are mini-inteins that lack a homing endonuclease. Both inteins can be overexpressed in Escherichia coli and purified as unspliced precursors; splicing can be induced in vitro by incubation with salt. However, one intein can splice in 0.5 M NaCl in vitro, whereas the other splices efficiently only in buffer containing over 2 M NaCl; the organism also requires high salt to grow, with the standard growth media containing over 3 M NaCl and about 0.75 M magnesium salts. Consistent with this difference in salt-dependent activity, an intein-containing precursor protein with both inteins promotes conditional alternative protein splicing (CAPS) to yield different spliced products dependent on the salt concentration. Native Trp fluorescence of the inteins suggests that the difference in activity may be due to partial unfolding of the inteins at lower salt concentrations. This differential salt sensitivity of intein activity may provide a useful mechanism for halophiles to respond to environmental changes.
Protein splicing is a self‐catalyzed, post‐translational process in which an intervening polypeptide sequence, or an intein, catalyzes its removal from two flanking polypeptide sequences, or exteins, as well as the ligation of the exteins. We are interested in the splicing capabilities of two inteins that interrupt the cdc21 protein from Haloquadratum walsbyi (Hwa). Hwa is a halophilic archaeon found in high salt environments such as the Red Sea. For this reason, we hypothesize that protein splicing may be salt dependent. We are interested in studying the splicing capabilities of both inteins separately and when expressed as part of the same protein. One intein, Cdc21a, does not splice on overexpression in E. coli, but can be induced to splice in vitro on incubation with 2.5 M sodium chloride at 28°. The other intein, Cdc21d, splices in vivo, and can be induced to splice further with higher concentrations of salt. When expressed in the same precursor fusion protein, the inteins continue to show differential activity conditional on salt concentration, leading to alternative spliced products. We hypothesize that this differential protein splicing is due to the folding of the protein under different salt conditions.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089), the Dreyfus Foundation (KVM), and Diane D. Brink P12 to the Alumni/Parent Summer Research Scholarship Fund. ALThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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