Purification of Proteins Fused to Either the Amino or Carboxy Terminus of the
            <i>Mycobacterium xenopi</i>
            Gyrase A Intein

Southworth, Maurice W.; Amaya, Kensey R.; Evans, Thomas C.; Xu, Ming‐Qun; Perler, Francine B.

doi:10.2144/99271st04

Cited by 224 publications

(199 citation statements)

References 3 publications

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“…Importantly, this mutation does not affect the N-terminal splicing reaction (22), which is the focus of this study, and for clarity is referred to as the wild-type sequence hereafter. For construction of the plasmid pTrcHis-Xa-GyrA wt , which encodes a factor Xa cleavage sequence between a poly(His) tag and the intein, the appropriate PCR product was cloned into the pTrcHisA vector (Invitrogen) by using BamHI and HindIII restriction sites.…”

Section: Methodsmentioning

confidence: 99%

Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Romanelli

Shekhtman

Cowburn

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

157

128

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Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The 1 JNC coupling constant for the (؊1) scissile peptide bond at the N-extein-intein junction was found to be Ϸ12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (؊1) scissile bond in its unusual conformation. Overall, these studies support the ''ground-state destabilization'' model as part of the mechanism of catalysis.

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Section: Methodsmentioning

confidence: 99%

Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Romanelli

Shekhtman

Cowburn

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

157

128

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“…In support of this structural inference, this amide NH within the C-extein is necessary for efficient intein-succinimide formation (16), suggesting that a defined branched extein conformation is important for splicing, perhaps by reducing the entropy of this region. Stabilization of this extein structure by a backbone interaction, as opposed to side-chain interactions, is attractive given the known promiscuity of inteins with respect to flanking extein residues, particularly N-extein residues (17,18). Further to that point, we note that Tyr side-chain at the −1 position in N-extein projects straight into solvent, whereas the side-chains of the remaining three N-extein residues lacked defined electron density and could not be modeled, presumably because of the absence of defined conformations.…”

Section: Significancementioning

confidence: 99%

Structure of the branched intermediate in protein splicing

Liu

Frutos

Bick

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use proteinengineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogenbond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site.expressed | protein semisynthesis P rotein splicing is a posttranslational modification in which an internal domain, termed an intein, excises itself from a host protein with concomitant ligation of the flanking sequences (termed the N-and C-exteins) (1, 2). Inteins are found in unicellular organisms from all domains of life (3) and belong to the HINT (Hedgehog INTein) superfamily of autoprocessing domains (4), which includes the cholesterol ligase domain found in the eponymous hedgehog family of developmental proteins present in all bilaterian animals (5, 6). High-resolution structures of inteins reveal a characteristic horseshoe-like β-sheet fold (7), common to all HINT family members (8), which positions catalytic residues from four conserved sequence blocks (A, B, F, G) in proximity to N-and C-terminal splice junctions (Fig. 1A). This structural information has aided mechanistic studies into the protein splicing process, which we know to be a multistep cascade involving a series of acyl-transfer reactions (Fig. 1B) (1, 2). Although a biological role for protein splicing remains elusive, an ever-deepening understanding of the splicing mechanism has led to the development of a wide range of biotechnology and chemical biology approaches based on engineered inteins (9).The most intriguing chemical step in protein splicing is inteinsuccinimide formation. This acts as the rate-limiting step in the process and leads to the resolution of the so-called branched (thio) ester intermediate species (Fig. 1B, step 3). This step involves nucleophilic attack of the side-chain primary amide group of a conserved Asn residue (the block G Asn) on the adjacent peptide bond (+1 scissile amide), leading to cleavage of the intein from the Cextein. This is an extre...

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“…5, B and C). Previously described protein purification systems that utilize N-terminal cleavage without the use of added thiols either undergo significant N-terminal cleavage during initial purification (33) or depend on the identity of the N-1 amino acid (34,35).…”

Section: ϫ6mentioning

confidence: 99%

Protein Splicing of a Pyrococcus abyssi Intein with a C-terminal Glutamine

Mills

Manning

Garcı́a

et al. 2004

Journal of Biological Chemistry

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Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond. Most reported inteins have a C-terminal asparagine residue, and it has been shown that cyclization of this residue is coupled to peptide bond cleavage between the intein and C-extein. We show that the intein interrupting the DNA polymerase II DP2 subunit in Pyrococcus abyssi, which has a C-terminal glutamine, is capable of facilitating protein splicing. Substitution of an asparagine for the C-terminal glutamine moderately improves the rate and extent of protein splicing. However, substitution of an alanine for the penultimate histidine residue, with either asparagine or glutamine in the C-terminal position, prevents protein splicing and facilitates cleavage at the intein N terminus. The intein facilitates in vitro protein splicing only at temperatures above 30°C and can be purified as a nonspliced precursor. This temperature dependence has enabled us to characterize the optimal in vitro splicing conditions and determine the rate constants for splicing as a function of temperature.Protein splicing is a post-translational auto-processing event by which an intervening polypeptide sequence, the intein, facilitates both its own excision from the flanking polypeptides, or exteins, as well as the ligation of the these segments (reviewed in Ref. 1). All of the catalytic groups required for protein splicing have been shown to lie within the intein and the two flanking amino acids (1).The splicing mechanism of the majority of inteins has been described in detail (1). The first step of splicing involves an N-O or N-S acyl rearrangement of the peptide bond at the intein N terminus to an ester or thioester (Fig. 1, step A). The second step is a transesterification between the nucleophilic residue at the N terminus of the C-extein 1 (Cys, Ser, or Thr) and the newly formed ester or thioester at the intein N terminus (Fig.

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Purification of Proteins Fused to Either the Amino or Carboxy Terminus of the Mycobacterium xenopi Gyrase A Intein

Cited by 224 publications

References 3 publications

Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Structure of the branched intermediate in protein splicing

Protein Splicing of a Pyrococcus abyssi Intein with a C-terminal Glutamine

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