Photomodulation of Protein Trans‐Splicing Through Backbone Photocaging of the DnaE Split Intein

Abstract

A novel strategy to modulate the assembly and trans-splicing activity of the Ssp DnaE split-intein was achieved by introducing two photolabile protecting groups onto the backbone of the C-intein polypeptide. This modification was not only able to efficiently block the trans-splicing activity, but also reduce significantly the binding affinity constant between the C-and N-intein fragments. The original activity of the wild-type split intein could be fully recovered by brief exposure to UV light. KeywordsBiotechnology; Proteins; Peptides; Photolysis; Recognition Intein-mediated protein splicing is a naturally occurring self-processing event in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond.[1-4] Protein splicing can be found to occur in cis or in trans. [5][6][7] In protein trans-splicing the intein self-processing domain is split in two fragments, called N-intein (I N ) and C-intein (I C ), respectively. These two intein fragments are inactive individually. However, they can bind each other with high specificity under appropriate conditions to form a functional protein-splicing domain.Since it was initially discovered, intein-mediated protein splicing and trans-splicing have been used in a multitude of applications ranging from protein purification, [8] protein backbone cyclization,[9-12] addition of labeled tags or other moieties, [13,14] protein immobilization, [15][16][17][18] protein semi-synthesis, [7] and segmental labeling or modification. [19][20][21][22] Of particular interest is the use of conditional protein trans-splicing, [23,24] which allows controlling the activity of a particular protein by modulating the assembly of the split intein moieties and therefore its splicing activity (Fig. 1).Recent work by Muir and co-workers has shown that the use of a reversible O-acyl backbone modification on the Ssp DnaE I C can be used to modify the splicing activity of this split-intein.[25] However, the lack of trans-splicing activity associated with the different O-acyl C-intein analogues was shown to be due to the introduction of local perturbations in the active site of

In-Cell Fluorescence Activation and Labeling of Proteins Mediated by FRET-Quenched Split Inteins

“…The fluorescein group was introduced at the C-terminus of the first four residues (Cys-Phe-Asn-Lys) of the C-extein, which are required for efficient trans-splicing. 21,27,45 The dabcyl group was first introduced at the N-terminus of the I C polypeptide (Q N , Fig. 2).…”

Recent progress in intein research: from mechanism to directed evolution and applications

“…Liberating a free N-terminus by light-irradiation (k 365nm ) restores split intein association and C-cleavage activity the backbone amides of Gly19 and Gly31 in the Ssp DnaE I C intein fragment the affinity to the I N fragment was reduced by *50 times and splicing was effectively inhibited. Splicing with purified proteins could then be restored by irradiation with light (k 365nm ) to wild-type levels with respect to both yield and kinetics [127] (Fig. 10a).…”

Protein trans-splicing as a protein ligation tool to study protein structure and function

“…This rapamycin-dependent PTS system has been shown to reconstitute proteins not only in test tubes, but also in live cells (83) and even whole organisms (84), allowing for control of protein function within a defined time period. Recently, light-inducible PTS systems have also been developed (85)(86)(87), which could be promising tools to activate protein function in vivo in a spatiotemporal manner using highly focused microscopic light sources.…”