Rational Design of an Improved Photo-Activatable Intein for the Production of Head-To-Tail Cyclized Peptides.

Rational Design of an Improved Photo-Activatable Intein for the Production of Head-To-Tail Cyclized Peptides.
Biol Chem. 2018 Nov 01;:
Authors: Böcker JK, Dörner W, Mootz HD
Abstract
Head-to-tail cyclization of genetically encoded peptides and proteins can be achieved with the SICLOPPS method by inserting the desired polypeptide between the C- and N-terminal fragments of a split intein. To prevent the intramolecular protein splicing reaction from spontaneously occurring upon folding of the intein domain, we have previously rendered this process light-dependent in a photo-controllable variant of the M86 intein, using genetically encoded ortho-nitrobenzyltyrosine at a structurally important position. Here we report improvements on this photo-intein with regard to expression yields and rate of cyclic peptide formation. The temporally defined photo-activation of the purified stable intein precursor enabled a kinetic analysis that identified the final resolution of the branched intermediate as the rate-determining individual reaction of the three steps catalyzed by the intein. With this knowledge, we prepared an R143H mutant with a block F histidine residue. This histidine is conserved in most inteins and helps catalyze the third step of succinimide formation. The engineered intein formed the cyclic peptide product up to 3-fold faster within the first 15 minutes after irradiation, underlining the potential of protein splicing pathway en...

Source: Biological Chemistry - November 9, 2018 Category: Chemistry Tags: Biol Chem Source Type: research