Falling Apart: the Self‐Catalyzed Process of Protein Splicing

Abstract

Protein splicing is a self‐catalyzed process where an intervening intein is excised, with the concomitant ligation of the two flanking exteins to form a mature protein. The inteins we studied interrupt the DNA Pol II of Pyrococcus abyssi and Pyrococcus horikoshii. These extremophile inteins conditionally splice at high temperatures. The Hyperthermophilic and Thermophilic Hairpin (HTH) is only found in the structures of archaeal inteins. We hypothesize that the HTH increases stability by elongating the central beta‐sheet, and thus mutations that disrupt this hairpin would affect the stability toward unfolding and the reactivity at the active site. To further understand the protein splicing of the P. abyssi intein, an intein with an Arg40Glu mutation in the HTH was expressed, and we studied its activity. We also purified 15‐N labeled intein for NMR (HSQC) analysis of residue‐level changes induced by the mutation. We have also purified a variant of the P. horikoshii PolII intein in order to attempt to solve the structure of an unspliced precursor intein.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.