“The relationship of structural stability to temperature‐dependent activity in a family of thermophilic inteins”

Abstract

Protein splicing is a post‐translational process by which a self‐catalyzing intervening sequence called an intein removes itself from a precursor protein and concomitantly ligates the flanking sequences, known as exteins. Our inteins of interest interrupt the DNA Polymerase II from the extreme thermophiles Thermococcus sibiricus (Tsi) and Thermococcus gammatolerans (Tga). We are interested in these inteins because of their sequence similarity to those from the extreme thermophiles Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho), all of which are mini‐inteins that lack a homing endonuclease domain. The Pho intein has been found to require higher temperature for splicing than the Pab intein, perhaps due to a shorter, more conformationally‐restricted region that replaces the missing homing endonuclease domain. The Tga and Tsi inteins have loop lengths more similar to the Pho intein. A splicing assay under increasing pressure will determine if there is a difference in activity in the two inteins that vary in the depths at which the parent organisms dwell. A preliminary assay at atmospheric pressure with an optimal isothermal temperature of 52°C showed that the Tga intein splices more efficiently than the Tsi intein. We will measure the temperature dependence of the intrinsic tryptophan fluorescence to link the overall stability of the fold to the difference in activity.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Camille and Henry Dreyfus Foundation (KVM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.