Live-cell protein engineering with an ultra-short split intein

Abstract

Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have not been utilized for the semisynthesis of proteins in live cells. Here, we biochemically and structurally characterize the naturally split intein VidaL. We show that this split intein, which features the shortest known N-terminal fragment, supports rapid and efficient proteintrans-splicing under a range of conditions, enabling semisynthesis of modified proteins both in vitro and in mammalian cells. The utility of this protein engineering system is illustrated through the traceless assembly of multidomain proteins whose biophysical properties render them incompatible with a single expression system, as well as by the semisynthesis of dual posttranslationally modified histone proteins in live cells. We also exploit the domain swapping function of VidaL to effect simultaneous modification and translocation of the nuclear protein HP1α in live cells. Collectively, our studies highlight the VidaL system as a tool for the precise chemical modification of cellular proteins with spatial and temporal control.

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

“…A S65A mutant ( 12 ) was still active in protein trans ‐splicing but its 4‐fold impaired rate demonstrates the importance of Ser65 (Figure 6C). Interestingly, the twisted arrangement at the N‐scissile bond, with the distortion leading to the coordination of the −1 carbonyl oxygen with N3 : 10 histidine, has only been observed so far in two intein structures [5d,18] and the present case is the first example for a class 3 intein. This is noteworthy because as a class 3 intein the AceL NrdHF intein employs the motif C2 thiol instead of the immediately adjacent side chain of the motif N1 : 1 residue at position 1 of the intein for the nucleophilic attack.…”

“…Another unusual sequence feature of the AceL NrdHF intein is the simultaneous absence of both catalytic histidine residues in the C2 and C1 motifs that usually contribute to the polarization of the asparagine side chain [19] and main chain carbonyl, [19,20] respectively, and thereby help in catalysis of asparagine cyclization and cleavage of the C-scissile bond. Introducing the motif C1 : 6 histidine by a G145H mutation (18) impaired all activity in the protein trans-splicing reaction ( Figure 8A and B). Structure-based mutagenesis simulations suggested a steric clash with the side chain of the N3 : 11 arginine Arg69, which is also found in hydrogen bonding distance (2.5 Å) to the Cscissile carbonyl oxygen and therefore might act in a compensatory mechanism in asparagine cyclization, as was shown for other inteins lacking the block C1 histidine.…”

The current chemical biology tool box for studying ion channels

“…This capability has been harnessed to assemble partial, non-functional Ca V and Na V channel fragments into full-length, functional channels using a single pair of split inteins (Subramanyam et al 2013;Lueck et al 2016). Given the increasing number of identified and splicing-optimized split inteins (Stevens et al 2016(Stevens et al , 2017Gramespacher et al 2018;Bhagawati et al 2019;Burton et al 2020;Pinto et al 2020), it is now also possible to use two orthogonal pairs of split inteins to insert a synthetic peptide between recombinantly expressed N-and C-terminal protein fragments (Fig. 5A).…”

“…2019; Burton et al . 2020; Pinto et al . 2020), it is now also possible to use two orthogonal pairs of split inteins to insert a synthetic peptide between recombinantly expressed N‐ and C‐terminal protein fragments (Fig.…”

Semisynthesis of functional transmembrane proteins in GUVs

“…[36][37][38][39] Split intein expressed protein ligation is advantageous in comparison to other protein semisynthesis strategies as it requires no additional chemical modifications after protein and peptide production. [39][40][41] Natural and engineered split inteins facilitate posttranslational protein trans splicing events and have been used for the determination of protein-protein interactions, 38,42 protein purification, 43 labeling of proteins with small molecules or unnatural amino acids, [44][45][46][47][48] segmental isotopic labeling for nuclear magnetic resonance (NMR), 40,[49][50][51][52] semisynthetic protein assembly in mammalian cells, 53,54 and cell membrane recruitment in expressed protein systems 55 (Figure S1). 37,56 Our strategy incorporates synthetic TM peptides directly in model cellular membranes, GUVs, 57,58 which bypasses the necessity of using cellular machinery to build and insert TM domains into a living cell membrane before reconstitution.…”