Segmental Isotope Labeling for Protein NMR Using Peptide Splicing

Yamazaki, Toshio; Otomo, Takanori; Oda, Natsuko; Kyōgoku, Yoshimasa; Uegaki, Koichi; Ito, Nobuyasu; Ishino, Yuji; Nakamura, Haruki

doi:10.1021/ja980776o

Cited by 226 publications

(172 citation statements)

References 13 publications

(12 reference statements)

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“…The Ssp DnaE intein may be the intein of choice for in vitro trans-splicing experiments as it has been demonstrated to undergo the trans-splicing reaction without the need for a denaturation/renaturation step, as was necessary with other inteins (21,23,24). Also, the addition of the CBD to either the N-or C-terminal intein fragment had no detectable inhibitory effect on splicing.…”

Section: Discussionmentioning

confidence: 99%

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Protein trans-Splicing and Cyclization by a Naturally Split Intein from the dnaE Gene ofSynechocystis Species PCC6803

Evans¹,

Martin²,

Kolly³

et al. 2000

Journal of Biological Chemistry

189

177

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A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro transsplicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the Nand C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.Protein splicing elements, termed inteins (1), catalyze their own excision from a primary translation product with the concomitant ligation of the flanking protein sequences (reviewed in Refs. 2-4). Inteins catalyze three highly coordinated reactions at the N-and C-terminal splice junctions (5, 6): 1) an acyl rearrangement at the N-terminal cysteine or serine; 2) a transesterification reaction between the two termini to form a branched ester or thioester intermediate; and 3) peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein. Inteins have been engineered to be versatile tools in protein purification (7-13), protein ligation (9, 10, 12, 14 -18), and in the formation of cyclic proteins and peptides (11,19,20). However, the ligation and cyclization approaches were limited by the need to generate an N-terminal cysteine and/or C-terminal thioester intermediate in vitro.In addition to inteins engineered to trans-splice (21-24), a naturally occurring split intein was recently identified in the dnaE gene encoding the catalytic subunit of DNA polymerase III of Synechocystis sp. PCC6803 (25). The N-terminal half of DnaE, followed by a 123-amino acid intein sequence, and the C-terminal half, preceded by a 36-amino acid intein sequence, are encoded by two open reading frames located more than 745 kilobases apart in the genome. When co-expressed in Escherichia coli, the two DnaE-intein fragments exhibited protein trans-splicing (25). In this report we have further investigated the cis-and trans-splicing activities of the Ssp DnaE intein in a foreign protein context. Furthermore, novel methods were developed that allow the on-column ligation of protein fragments as well as the in vivo and in vitro cyclization of p...

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Section: Discussionmentioning

confidence: 99%

“…In addition to inteins engineered to trans-splice (21)(22)(23)(24), a naturally occurring split intein was recently identified in the dnaE gene encoding the catalytic subunit of DNA polymerase III of Synechocystis sp. PCC6803 (25).…”

mentioning

confidence: 99%

Protein trans-Splicing and Cyclization by a Naturally Split Intein from the dnaE Gene ofSynechocystis Species PCC6803

Evans¹,

Martin²,

Kolly³

et al. 2000

Journal of Biological Chemistry

189

177

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“…Protein splicing is a self-catalytic reaction catalyzed by an intervening sequence in a host protein, termed an intein, which ligates the flanking protein sequences, termed exteins, by a peptide bond and concomitantly excises itself from the host protein [3]. Protein splicing in trans could ligate two foreign peptide chains that are fused with either N-or C-terminal fragments (N-or C-inteins) of a split intein [4][5][6]. Protein trans-splicing system has opened many applications including segmental isotopic labelling of proteins, protein cyclization, in vivo protein engineering, and site-specific chemical modifications [6][7][8][9][10][11][12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

“…Protein splicing in trans could ligate two foreign peptide chains that are fused with either N-or C-terminal fragments (N-or C-inteins) of a split intein [4][5][6]. Protein trans-splicing system has opened many applications including segmental isotopic labelling of proteins, protein cyclization, in vivo protein engineering, and site-specific chemical modifications [6][7][8][9][10][11][12][13][14][15]. However, protein ligation by protein trans-splicing can be significantly modulated by the junction sequences as well as by the extein sequences, which could limit the applications of protein trans-splicing [16].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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a b s t r a c tNaturally 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.

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“…Yamazaki et al have applied trans-splicing to the segmental labeling of RNA polymerase subunit a by splitting an intein from Pyrococcus furiosus, PI-PfuI (Fig. 1.5 B) [70]. Each half of the split intein fused to the N-(or C)-extein was produced separately, one in isotopically labeled and the other in unlabeled medium.…”

Section: Reconstitution Of Split Inteinsmentioning

confidence: 99%

Modern Methods for the Expression of Proteins in Isotopically Enriched Form

Patzelt

Goto

Iwaï

et al. 2002

BioNMR in Drug Research

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The introduction of stable isotopes into proteins has significantly reduced the time requirements for structure elucidation of biomolecules. Moreover, structural studies of proteins with molecular weights exceeding the 10 kDa limit are usually not possible without uniform isotope labeling because of severe resonance overlap and inefficient coherence transfer along the rather small 3 J 1 H-1 H couplings. Nowadays, efficient expression of recombinant proteins is a prerequisite for many techniques used in structural biology, but the requirement for isotope labeling in particular often precludes NMR (nuclear magnetic resonance) structure determination of proteins isolated from natural sources. Specifically, proteins that have been uniformly labeled with 15 N and/or 13 C are commonly required for NMR spectroscopy, especially for backbone chemical shift assignment procedures, which are greatly facilitated by the use of a series of rather sensitive multi-dimensional triple-resonance NMR experiments (see Chapt. 4) [1], in a process that can also be automated with good success [2]. Moreover, the random replacement of nonexchangeable protons by deuterons reduces 1 H-1 H dipolar interactions and scalar couplings, thereby reducing peak line widths considerably and allowing structure elucidation of proteins exceeding 30 kDa [3,4]. Random fractionally deuterated protein samples also permit the use of longer mixing times in NOESY (nuclear Overhauser effect spectroscopy) experiments, since spin-diffusion pathways are largely eliminated. In addition, transverse-relaxation optimized spectroscopy (TROSY [5], see also Chapt. 10), which has been used for molecules larger than 100 kDa [6], benefits dramatically from deuteration. This stems from the fact that the TROSY component that is narrowed by the DD-CSA compensation is broadened by dipolar interactions with nearby protons.Besides uniform labeling approaches, stable isotopes can also be introduced at specific sites in proteins in order to simplify the assignment process and to isolate spectral information from the region of interest. For example, biosynthetically-directed fractional 13 C labeling offers the possibility of making stereospecific assignments of all isopropyl methyl groups of Val and Leu residues [7]. In another approach often used for solid-state NMR termed residue-specific labeling, isotope labels are introduced at single sites in a protein, as described in another chapter of this volume (Chapt. 11). A related scheme, called amino acid-type labeling, is accomplished by expression in an amino acid-based medium

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Segmental Isotope Labeling for Protein NMR Using Peptide Splicing

Cited by 226 publications

References 13 publications

Protein trans-Splicing and Cyclization by a Naturally Split Intein from the dnaE Gene ofSynechocystis Species PCC6803

Protein trans-Splicing and Cyclization by a Naturally Split Intein from the dnaE Gene ofSynechocystis Species PCC6803

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

Modern Methods for the Expression of Proteins in Isotopically Enriched Form

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