A. Sesilja Aranko

Segmental isotopic labeling of multi-domain and fusion proteins by protein trans -splicing in vivo and in vitro

Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or α-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7–13 d depending on the protocol used.

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification

Naturally split DnaE intein from Nostoc punctiforme (Npu) has robust protein trans‐splicing activity and high tolerance of sequence variations at the splicing junctions. We determined the solution structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C‐terminal half (C‐intein) composed of six residues. In vivo and in vitro protein ligation of model proteins by the newly designed split intein were demonstrated.

In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins.

Background Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized. Principal Findings We report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents. Conclusion The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing.

Segmental Isotopic Labeling of a Central Domain in a Multidomain Protein by Protein Trans-Splicing Using Only One Robust DnaE Intein†

Many proteins, including cellular signaling proteins, cellsurface receptors, and modular enzymes, are constructed from individual domains and connecting linkers. Structural analysis by NMR spectroscopy or by X-ray crystallography often focuses on self-contained domains excised from full-length proteins so as to reduce the molecular weight to a manageable size for high-quality NMR spectra or to improve crystallization by removing disordered regions. Such minimization often neglects functional aspects of a domain in an intact protein and may not represent all aspects of the structure– function relationship in the full-length context. While the structure determination of individual, isolated domains has been tremendously accelerated—in part through several structural genomics consortia—understanding of domain– domain interactions within a multidomain protein is lacking behind. NMR spectroscopy is an ideal tool for characterizing such, often transient, interactions. However, NMR spectroscopic analysis of large proteins often suffers from signal overlap. This problem becomes even more profound when proteins contain recurring modular domains and/or highly disordered regions. One solution to this overlap problem is segmental isotopic labeling that enables the exclusive incorporation of NMR active, stable isotopes into selected domains or parts of larger proteins, thereby reducing the complexity of the NMR spectra. In the past years, we have advanced segmental isotopic labeling procedures by discovering and engineering robust protein splicing domains (inteins) and by developing in vivo methods with the time-delayed dual expression system. While segmental isotopic labeling of Nor Cterminal halves of proteins by protein ligation of two fragments has become a well-established routine procedure, labeling of a middle domain or segment in a larger protein still poses significant challenges. Native chemical ligation (NCL) or expressed protein ligation (EPL) could be used for such a three-fragment ligation, however, the requirement for isotope-enriched peptides with a protected N-terminal cysteine residue is not cost-effective and the protection/deprotection steps to create a reactive N-terminal cysteine could be laborintensive. One promising approach to ligate three polypeptide fragments is to use protein trans-splicing (PTS) with two split inteins (denoted as Int1 and Int2) because PTS does not require any cumbersome steps to create an N-terminal cysteine needed for EPL (Figure 1a). 10, 11] For this approach, however, the two split inteins must be orthogonal without any cross-reactivity and have high splicing efficiency. Segmental labeling of a central region of a protein by three-fragment ligation was elegantly realized with the two orthogonal split inteins, PI-PfuI and PI-PfuII. However, the artificially split inteins of PI-PfuI and PI-PfuII required tedious refolding and optimization steps to restore the splicing activity because the fusion proteins with the split intein fragments were insoluble. 12] Since refolding can also result in inactive proteins, avoiding any refolding steps would be highly beneficial. This can be achieved with naturally occurring split inteins, such as DnaE inteins from cyanobacteria, which usually do not require any refolding step to induce PTS. 6, 8, 13,14] Particularly, DnaE intein from Nostoc punctiforme (Npu) could be ideally suited based on the combination of its robust splicing activity, high tolerance of sequence variations at the splicing junctions, and its high solubility. For three-fragment ligation, however, the DnaE inteins cannot be directly used because the domain or fragment inserted in the middle of the precursor protein containing both Nand C-intein (IN/IC) could result in cyclization or polymerization (Figure 1b). 14, 15] Herein, we develop a novel strategy for three-fragment protein ligation that overcomes the cyclization/polymerization problem but still utilizes only one robust DnaE intein to achieve higher yields. To circumvent the inevitable cyclization using only one split intein (Figure 1b), we exploited engineered NpuDnaE inteins with the same sequence but different split sites (Figure 1c). Shortening, [*] A. S. Aranko, Dr. H. Iwa Research Program in Structural Biology and Biophysics Institute of Biotechnology, University of Helsinki P.O. Box 65, Helsinki 00014 (Finland) Fax: (+ 358)9-1915-9541 E-mail: hideo.iwai@helsinki.fi Homepage: http://www.biocenter.helsinki.fi/bi/iwai/ A. E. L. Busche, Dr. M. Talebzadeh-Farooji, Dr. F. Bernhard, Prof. Dr. V. D tsch Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance and Cluster of Excellence Frankfurt Macromolecular Complexes (CEF), University of Frankfurt 60438 Frankfurt (Germany) [] These authors contributed equally to this work.

Segmental Isotopic Labelling of a Multidomain Protein by Protein Ligation by Protein Trans-Splicing

Segmental isotopic labelling is a powerful method for the incorporation of stable isotopes into particular regions within proteins for NMR detection, thereby reducing the complexity of NMR spectra and offering the potential to perform sequential assignments. Here we have demonstrated segmental isotopic labelling of a domain in a multidomain protein both in vivo and in vitro through protein ligation by protein trans-splicing. This robust protein trans-splicing approach could open possibilities for studying particular domains in intact proteins without dissection into smaller globular domains. Recent advances in optimization of transverse-relaxation in NMR spectroscopy have opened avenues for study of larger molecules (close to 1 MDa). However, sequential resonance assignments in large proteins remain time-consuming and challenging because of the increased number of signals and signal overlapping. Segmental isotopic labelling is one promising approach among numerous isotope-labelling techniques, because, unlike in the case of selective amino acid labelling, segmentally isotope-labelled samples can be directly analysed by tripleresonance NMR techniques developed for sequential resonance assignments. Segmentally isotope-labelled proteins have been prepared either by expressed protein ligation (EPL), which makes use of native chemical ligation (NCL), 6] or by protein trans-splicing (PTS), through the use of artificially split protein splicing domains (inteins). 7] EPL requires the preparation of an a-thioester group from a thiol reagent and an N-terminal cysteine residue by proteolysis in vitro (Scheme 1A), which demands considerable preparation efforts, 8] although an easier approach has recently been proposed. In protein splicing, an intein catalyses protein ligation of two polypeptide fragments fused to the Nand C-terminal ends of an intein. Protein splicing could take place in trans, when an intein is split into two fragments (Scheme 1B). 10] Segmental isotopic labelling through protein trans-splicing with artificially split inteins requires no additional thiol reagent nor cofactor, but denaturation and renaturation steps are necessary before protein-splicing activity can be restored. Unlike artificially split inteins, naturally split inteins do not require any denaturation and renaturation steps for protein splicing. Therefore, these have been suggested as potentially useful for segmental isotopic labelling of multidomain proteins. Protein trans-splicing with naturally split inteins has advantages over EPL because protein ligation can be performed not only in vitro but also in vivo, making it possible to achieve segmental isotopic labelling in vivo. Despite its many potential applications, it has never been used for segmental isotopic labelling of multidomain proteins except for a fusion tag for enhancing protein solubility. This is because the protein-splicing activity of the split inteins could be negatively affected even when naturally split inteins were fused with the Scheme 1. Protein ligation by A) expressed protein ligation and B) protein trans-splicing. C) Outline of the in vivo procedure for segmental isotopic labelling used in this article.

Intermolecular domain swapping induces intein-mediated protein alternative splicing.

Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms. We now report protein alternative splicing, in which new protein sequences can be produced by protein recombination by intermolecular domain swapping of inteins, as elucidated by NMR spectroscopy and crystal structures. We demonstrate that intein-mediated protein alternative splicing could be a new strategy to increase protein diversity (that is, functions) without any modification in genetic backgrounds. We also exploited it as a post-translational protein conformation-driven switch of protein functions (for example, as highly specific protein interference).

Nature's recipe for splitting inteins

Protein splicing in trans by split inteins has increasingly become a powerful protein-engineering tool for protein ligation, both in vivo and in vitro. Over 100 naturally occurring and artificially engineered split inteins have been reported for protein ligation using protein trans-splicing. Here, we review the current status of the reported split inteins in order to delineate an empirical or rational strategy for constructing new split inteins suitable for various applications in biotechnology and chemical biology.

Structural basis for protein trans‐splicing by a bacterial intein‐like domain – protein ligation without nucleophilic side chains

Protein splicing in trans by split inteins has become a useful tool for protein engineering in vivo and in vitro. Inteins require Cys, Ser or Thr at the first residue of the C‐terminal flanking sequence because a thiol or hydroxyl group in the side chains is a nucleophile indispensable for the trans‐esterification step during protein splicing. Newly‐identified distinct sequences with homology to the hedgehog/intein superfamily, called bacterial intein‐like (BIL) domains, often do not have Cys, Ser, or Thr as the obligatory nucleophilic residue found in inteins. We demonstrated that BIL domains from Clostridium thermocellum (Cth) are proficient at protein splicing without any sequence changes. We determined the first solution NMR structure of a BIL domain, CthBIL4, to guide engineering of split BIL domains for protein ligation. The newly‐engineered split BIL domain could catalyze protein ligation by trans‐splicing. Protein ligation without any nucleophilic residues of Cys, Ser and Thr could alleviate junction sequence requirements for protein trans‐splicing imposed by split inteins and could broaden applications of protein ligation by protein trans‐splicing.

Use of protein trans‐splicing to produce active and segmentally 2H, 15N labeled mannuronan C5‐epimerase AlgE4

Alginate epimerases are large multidomain proteins capable of epimerising C5 on β‐D‐mannuronic acid (M) turning it into α‐L‐guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution‐state NMR are hampered by their size—the smallest epimerase, AlgE4, consisting of one A‐ and one R‐module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally 2H, 15N labeled AlgE4 isotopomeres (A‐[2H, 15N]‐R and [2H, 15N]‐A‐R) by protein trans‐splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild‐type AlgE4.

Salt-inducible Protein Splicing in cis and trans by Inteins from Extremely Halophilic Archaea as a Novel Protein-Engineering Tool.

Intervening protein sequences (inteins) from extremely halophilic haloarchaea can be inactive under low salinity but could be activated by increasing the salt content to a specific concentration for each intein. The halo-obligatory inteins confer high solubility under both low and high salinity conditions. We showed the broad utility of salt-dependent protein splicing in cis and trans by demonstrating backbone cyclization, self-cleavage for purification, and scarless protein ligation for segmental isotopic labeling. Artificially split MCM2 intein derived from Halorhabdus utahensis remained highly soluble and was capable of protein trans-splicing with excellent ligation kinetics by reassembly under high salinity conditions. Importantly, the MCM2 intein has the active site residue of Ser at the +1 position, which remains in the ligated product, instead of Cys as found in many other efficient split inteins. Since Ser is more abundant than Cys in proteins, the novel split intein could widen the applications of segmental labeling in protein NMR spectroscopy and traceless protein ligation by exploiting a Ser residue in the native sequences as the +1 position of the MCM2 intein. The split halo-obligatory intein was successfully used to demonstrate the utility in NMR investigation of intact proteins by producing segmentally isotope-labeled intact TonB protein from Helicobacter pylori.