Kinetic Control of One‐Pot Trans‐Splicing Reactions by Using a Wild‐Type and Designed Split Intein

Abstract

Keywords ion pairs; protein design; protein-protein interactions; semisynthesisCoulombic forces play an important role in facilitating protein-protein interactions. It has been demonstrated that a strong electrostatic potential between two interacting proteins correlates with a fast rate of association and a strong binding affinity. [1] Indeed, this principle has been exploited to enhance the binding properties of engineered protein interfaces. [2] Nonetheless, little research has focused on utilizing intermolecular ion pairs to modulate specificity in protein-protein interactions. [3][4][5] Naturally split inteins are a potentially interesting system for engineering electrostatically driven specificity, as the formation of a catalytically competent structure requires the association of two oppositely charged protomers. In their endogenous environment, split inteins catalyze protein transsplicing (PTS, Scheme 1) of essential gene fragments, and thus are under evolutionary pressure to associate rapidly with high fidelity and dissociate slowly post-catalysis to prevent the re-formation of unproductive complexes. Out of their native context, split inteins have seen widespread use in a number of biotechnological applications, as a result of their capacity to ligate flanking protein sequences (exteins) in trans. [6] Applications of PTS include segmental isotopic labeling of proteins for NMR spectroscopy, [7] protein immobilization, [8] the labeling of proteins with extrinsic probes, [9] protein and peptide cyclization, [10] and control of protein function. [11,12] Despite the utility of PTS, little is known about what drives efficient association of split intein fragments. Sequence alignments of naturally split inteins show highly conserved charge segregation, with acidic residues concentrated at specific positions on the N-intein and basic residues conserved on the C-intein (N-and C-terminal protomers, Figure 1a). [13] Furthermore, a bioinformatic sequence analysis of the intein family indicates that this charge segregation is significantly more prevalent in naturally split inteins than intact ones ( Figure 1c). Interestingly, when the conserved charged residues are mapped onto the structure determined by NMR spectrocopy of the wild-type DnaE intein from Nostoc punctiforme (Npu WT ), many are found to be participating in intermolecular ion pairs and ion triads (Figure 1b).

The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from acis-splicing intein

“…This problem has been alleviated in part by salt-inducible split inteins derived from a salt-inducible intein from extreme halophilic archaea, but yet requiring salt-induction for transsplicing 40 . Having more than two robust split inteins which are not cross-active (orthogonal) could widen the applications of PTS because two orthogonal split inteins enable us to perform three-fragment ligation efficiently 20,21,22,23 . Naturally split DnaE inteins such as NpuDnaE intein, however, are cross-active to other naturally occurring split DnaE inteins, despite its robust splicing activity and high tolerance of variations at the splice junctions 29,32,41 .…”

“…Previously, it has been suggested that local charge distributions between naturally split intein halves are important for their association 18,23,29 . We observed extensive charge-charge interactions in the crystal structure of the gp41-1 intein, as observed among other naturally split inteins 29 .…”

“…For such multi-fragment applications, two non-cross active (orthogonal) split inteins are required in order to suppress undesired cross-activity. Several approaches have been used to circumvent the crossreactivity, such as utilizing different split sites of the NpuDnaE intein or kinetic control of two split inteins 21,22,23 . The three-dimensional structure of the gp41-1 intein revealed charge distributions different from the NpuDnaE intein in the corresponding β3 and β6 strands.…”

“…Despite increasing interests in the utilization of various split inteins for protein engineering purposes, the repertoire of split inteins with both robust protein splicing activity and high sequence tolerance at the splice junctions are still small. Particularly, pairs of orthogonal split inteins are desirable for one-pot multiple fragment protein ligation by PTS requiring two orthogonal split inteins 20,21,22,23 . Previous engineering attempts to derive novel split inteins from naturally occurring cis-splicing inteins did not result in highly robust split 3 inteins, indicating that cis-splicing inteins are not optimized for trans-splicing, unlike naturally occurring split inteins 15,24,25 .…”

Circular zymogens of human ribonuclease 1

“…25 The NpuN and NpuC intein fragments were obtained from vectors provided by Tom W. Muir (Princeton University). 28 DNA oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA).…”