Inteins catalyze a post-translational modification known as protein splicing, where the intein removes itself from a precursor protein and concomitantly ligates the flanking protein sequences with a peptide bond. Over the past two decades, inteins have risen from a peculiarity to a rich source of applications in biotechnology, biomedicine, and protein chemistry. In this review, we focus on developments of intein-related research spanning the last 5 years, including the three different splicing mechanisms and their molecular underpinnings, the directed evolution of inteins towards improved splicing in exogenous protein contexts, as well as novel applications of inteins for cell biology and protein engineering, which were made possible by a clearer understanding of the protein splicing mechanism.

Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine-thioester and phenylalanine-AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.

Obtaining insights into the molecular structure and dynamics of a protein by NMR spectroscopy and other in-solution biophysical methods relies heavily on the incorporation of isotopic labels or other chemical modifications such as fluorescent groups into the protein of interest. These types of modifications can be elegantly achieved with the use of split inteins in a site- and/or region-specific manner. Split inteins are split derivatives of the protein splicing element intein, and catalyze the formation of a peptide bond between two proteins. Recent progress in split intein engineering provided the opportunity to also perform peptide bond formation between a protein and a chemically synthesized peptide. We review the current state-of-the-art in preparing segmental isotope-labeled proteins for NMR spectroscopy, and highlight the importance of split intein orthogonality for the ligation of a protein from multiple fragments. Furthermore, we use split intein-mediated site-specific fluorescent labeling as a framework to illustrate the general usefulness of split inteins for custom protein modifications in the realm of structural biology. We also address some limitations of split intein technology, and offer constructive advice to overcome these shortcomings.

Conventional split inteins have been useful for trans-splicing between recombinant proteins, and an artificial S1 split intein is useful for adding synthetic peptide onto the N terminus of recombinant proteins. Here we have engineered a novel S11 split intein for trans-splicing synthetic peptide onto the C terminus of recombinant proteins. The C-intein of the S11 split intein is extremely small (6 amino acids (aa)); thus it can easily be produced together with a synthetic C-extein to be added to the C terminus of target proteins. The S11 intein was derived from the Ssp GyrB intein after deleting the homing endonuclease domain and splitting the remaining intein sequence near the C terminus, producing a 150-aa N-intein (I N ) and a 6-aa C-intein (I C ). Its trans-splicing activity was demonstrated first in Escherichia coli cells and then in vitro for trans-splicing between a synthetic peptide and a recombinant protein. The in vitro trans-splicing reaction exhibited a typical rate constant of (6.9 ؎ 2.2) ؋ 10 ؊5 s ؊1 and reached a high efficiency of ϳ80%. This S11 split intein can be useful for adding any desirable chemical groups to the C terminus of a protein of interest, which may include modified and unnatural amino acids, biotin and fluorescent labels, and even drug molecules.Inteins are internal protein sequences and catalyze a proteinsplicing reaction, which precisely excises the intein sequence and join the flanking sequences (N-and C-exteins) with a peptide bond (1). The reaction mechanism of protein splicing has been well studied (2, 3), and the conserved crystal structure of the protein-splicing domain of inteins consists of ϳ12 ␤-strands that form a disk-like compact structure with the two splicing junctions located in a central cleft (4 -7). More than 400 inteins and intein-like sequences have been found in a wide variety of host proteins and in microorganisms including bacteria, Archaea, and eukaryotes (8, 9). Inteins are typically 350 -550 aa 3 in size (8, 9) with the majority containing a homing endonuclease domain. Some inteins are as large as 1650 aa in size and contain tandem repeats (10, 11). Inteins can lose the endonuclease domain, and the resulting functional mini-intein retains only the splicing domain ϳ140 aa in size (12-14).Split inteins are essentially mini-inteins broken into two pieces and able to reassociate and carry out protein trans-splicing (15). In certain cyanobacteria, a natural split intein is responsible for producing a mature DnaE protein by transsplicing two separate polypeptides expressed from two separate genes (16,17). Artificial split inteins have also been engineered by splitting the sequences of contiguous inteins to resemble naturally occurring split inteins (13,18,19). Split inteins have many practical uses including the production of recombinant proteins from fragments and the circularization of recombinant proteins (20). However, conventional split inteins are less useful for adding synthetic peptide to proteins because their split sites are relatively close to the...

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