Protein splicing of a Pyrococcus abyssi intein with a C-terminal glutamine.
Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond. Most reported inteins have a C-terminal asparagine residue, and it has been shown that cyclization of this residue is coupled to peptide bond cleavage between the intein and C-extein. We show that the intein interrupting the DNA polymerase II DP2 subunit in Pyrococcus abyssi, which has a C-terminal glutamine, is capable of facilitating protein splicing. Substitution of an asparagine for the C-terminal glutamine moderately improves the rate and extent of protein splicing. However, substitution of an alanine for the penultimate histidine residue, with either asparagine or glutamine in the C-terminal position, prevents protein splicing and facilitates cleavage at the intein N terminus. The intein facilitates in vitro protein splicing only at temperatures above 30 degrees C and can be purified as a nonspliced precursor. This temperature dependence has enabled us to characterize the optimal in vitro splicing conditions and determine the rate constants for splicing as a function of temperature.
Mills
Kenneth V
KV
College of the Holy Cross, Department of Chemistry, 1 College Street, Worcester, MA 01610, USA. kmills@holycross.edu
Manning
Jennifer S
JS
Garcia
Alicia M
AM
Wuerdeman
Lisa A
LA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004257:DNA Polymerase II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D005973:Glutamine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019154:Protein Splicing / Q000235:genetics*; D021122:Protein Subunits / Q000235:genetics; D043903:Pyrococcus abyssi / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D017931:DNA Primers; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D001216:Asparagine; D004257:DNA Polymerase II