Mutational analysis of protein splicing, cleavage, and self-association reactions mediated by the naturally split Ssp DnaE intein.
The ability to separately purify the naturally split Synechocystis sp. PCC6803 (Ssp) DnaE intein domains has allowed detailed examination of both universal and Ssp DnaE intein-specific steps in the protein splicing pathway. By engineering substitutions at both the +1 and penultimate intein positions, we have further characterized intein reaction kinetics in this system. Replacement of the crucial +1Cys with serine decreased N-terminal cleavage and trans-splicing rates; however, this substitution did not prevent splicing or the ability of ZnCl2 to inhibit it. Substitution of the penultimate intein residue (alanine) with a typically conserved histidine did not increase the rate or extent of trans-splicing or cleavage under typical assay conditions. Despite the observation that this histidine aids in asparagine cyclization for other inteins, it did not encourage C-terminal cleavage for the Ssp DnaE intein or uncouple it from N-terminal cleavage. Both the +1Ser and Ala to His mutants were insensitive to ZnCl2 during trans-cleavage experiments, uncoupling a previously linked inhibition in asparagine cyclization from an inhibition in trans-thioesterification detected for the wild-type intein.
Nichols
Nicole Magnasco
NM
New England Biolabs, Inc., Beverly, Massachusetts 01915-5599, USA.
Evans
Thomas C
TC
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001667:Binding, Competitive; D000458:Cyanobacteria / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D019154:Protein Splicing / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012694:Serine; D006639:Histidine; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D000409:Alanine