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J. Biol. Chem., Vol. 279, Issue 20, 20685-20691, May 14, 2004

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

Kenneth V. Mills, Jennifer S. Manning, Alicia M. Garcia, and Lisa A. Wuerdeman

From the College of the Holy Cross, Department of Chemistry, Worcester, Massachusetts 01610

Received for publication, January 27, 2004 , and in revised form, March 4, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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-extein¹ (Cys, Ser, or Thr) and the newly formed ester or thioester at the intein N terminus (Fig. 1, step B). Next, cyclization of the asparagine residue at the C terminus of the intein results in peptide bond cleavage and excision of the intein. Evidence in support of this step includes the isolation and analysis of C-terminal aminosuccinimide residues formed during protein splicing mediated by the Pyrococcus sp. GB-D Pol intein (2, 3) and the Saccharomyces cerevisiae VMA intein (4). In addition, branched intermediate accumulates when the conserved C-terminal Asn is mutated to Gln or Asp in the Pyrococcus sp. GB-D Pol intein (5), and splicing is prevented by these mutations in the S. cerevisiae VMA intein (6). Finally, the ester or thioester linking the extein segment is rearranged to a peptide bond (Fig. 1, step G), and the intein C-terminal aminosuccinimide residue may be hydrolyzed.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Proposed mechanism for protein splicing. In most inteins, protein splicing initiates via an N-S acyl rearrangement to form a linear thioester intermediate (step A), followed by a trans-thioesterification reaction to form a branched thioester (step B) (reviewed in Ref. 1). The ligation of the N- and C-extein segments by a thioester bond (step F, which is followed by step G to form a stable amide bond between extein segments) is likely coupled to one of three steps that results in cleavage of the intein-C-extein peptide bond in the P. abyssi PolII intein. Step C, cyclization of glutamine to form a C-terminal aminoglutarimide residue. Step D, cyclization of glutamine to form a C-terminal aminoglutaranhydride. Step E, attack of the N-terminal Cys residue to form a cyclic intein intermediate.

Inteins that follow alternate splicing mechanisms have recently been described. For example, inteins lacking an N-terminal nucleophile, in which Ala replaces the Cys or Ser at the intein N terminus, can facilitate splicing (7, 8). These inteins likely bypass the first step of splicing, although at least one has been shown to be capable of facilitating an N-S acyl rearrangement of the peptide bond linking the N-extein and the intein when the Ala at the intein N terminus is replaced with Cys (7).

There are also examples of variations of the third step of protein splicing, in which Asn cyclization is coupled to peptide bond cleavage. It was reported that a Gln replaces the highly conserved C-terminal Asn in the intein that interrupts the ribonucleotide reductase (RNR) of the Chilo iridescent virus (CIV) (9). This intein has been shown to be capable of facilitating protein splicing in Escherichia coli (10). The intein that interrupts the RNR of Carboxydothermus hydrogenoformus has an aspartic acid residue at its C terminus and is also splicing-competent (10). In addition, the C. hydrogenoformus RNR intein is capable of facilitating cleavage at the intein C terminus, even when the C-terminal residue is mutated to Ala, suggesting an alternate mechanism to side chain cyclization for C-terminal cleavage (10).

It has been postulated that the conserved penultimate His residue plays a role in Asn cyclization. Protein splicing is arrested, and branched intermediate accumulates when the penultimate His in the Pyrococcus sp. GB-D Pol intein is mutated to Ala (5). Protein splicing can be enhanced in inteins lacking a penultimate His by introducing a His via mutation (11, 12). Structural evidence also points to a role for the penultimate His in Asn cyclization. For instance, in the Mycobacterium xenopi GyrA intein crystal structure, His-197 is in a position to serve as a proton donor to the backbone amide nitrogen of the scissile bond (13). In the Synechocystis sp. PCC6803 DnaB intein crystal structure, the penultimate His serves as a proton donor to the carbonyl oxygen of the terminal Asn (14). However, the conserved penultimate His is not required to mediate efficient protein splicing in other inteins, because some inteins splice in its absence (7, 12, 15), and in some of these cases splicing is impaired when the penultimate residue is replaced with His (7, 12).

In this paper, we describe the temperature-dependent protein splicing of the intein interrupting the DNA polymerase II, DP2 subunit in Pyrococcus abyssi. The P. abyssi PolII intein is the second intein described that can facilitate protein splicing with a C-terminal glutamine residue. Because this intein can be purified as an unspliced precursor and induced to splice via an increase in temperature, we were able to compare the in vitro protein splicing rate and the efficiency of the intein with a series of C-terminal mutations.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
P. abyssi Growth—A culture of P. abyssi was kindly provided by Prof. Frank Robb (Center for Marine Biotechnology, University of Maryland Biotechnology Institute). A 2% innoculum was transferred to P. abyssi medium and incubated under nitrogen at 95 °C for 14 h. Sulfur was removed by centrifugation at 500 x g in a Beckman J2–21 centrifuge for 5 min. The supernatant was transferred to a clean tube, and the cells were pelleted at 5000 x g for 20 min.

P. abyssi medium was prepared essentially as described (16) and consisted of 0.25 g/liter NH₄Cl, 0.05 g/liter CaCl₂, 0.05 g/liter Na₂HPO₄, 5 g/liter KCl, 3.45 g/liter MgSO₄, 6 g/liter piperazine-1,4-bis(2-ethanesulfonic acid), 20 g/liter NaCl, 0.5 g/liter cysteine, 0.5 g/liter cysteine, 5 g/liter yeast extract, 100 mg/liter pyridoxine HCl, 50 mg/liter calcium pantothenate, 50 mg/liter nicotinic acid, 50 mg/liter p-aminobenzoic acid, 50 mg/liter riboflavin, 50 mg/liter thiamin HCl, 50 mg/liter thioctic acid, 20 mg/liter folic acid, 20 mg/liter biotin, 1 mg/liter cyanocobalamine, and, after adjustment to pH 7.4 at 96 °C, 1 ml of 0.1% resazurin and about 20 g/liter of precipitated sulfur. The medium was steamed repeatedly in autoclave until yellow and transferred to capped serum bottles. Head gas was exchanged with nitrogen five to six times.

DNA Purification—The P. abyssi cell pellet was washed and resuspended in 10 ml of TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA). After the addition of 0.5 ml of 10% SDS and 50 µl of 20 mg/ml proteinase K, the cell suspension was incubated at 37 °C for 1 h. This was followed by the addition of 1.4 ml of 5 M NaCl and 1.5 ml of 10% hexadecyltrimethyl ammonium bromide in 0.7 M NaCl and incubation for 2 min at 65 °C. DNA was extracted first with 24:1 chloroform:isoamyl alcohol and then with 1:1 phenol:chloroform. To precipitate the DNA, 0.6 volumes of isopropanol were added, and the sample was centrifuged at 14,000 x g for 20 min to pellet the DNA, let air dry, and resuspended in 100 µl of deionized water.

Plasmid Preparation—Plasmid pPabPol1 encodes PabPol1, an inframe fusion of E. coli MBP with the seven C-terminal residues of the P. abyssi PolII N-extein and the first 179 residues of the intein. To create this plasmid, the intein gene was amplified from genomic DNA via PCR using oligonucleotide primers P1U (5'-TACGCTAGGCCTTACATGCATGCT) and P1L (5'-TTGATGCGTTAGAATATTTTCATTTAT). (All of the oligonucleotides were purchased from MWG Biotech, High Point, NC.) The PCR product was digested with StuI and XmnI and ligated into the XmnI site of plasmid pMal-c2x (New England Biolabs, Beverly, MA) to give plasmid pPabPol1.

To introduce a C-terminal poly-His tag as the C-extein, plasmid pPabPol1His was constructed. Plasmid pPabPol1His encodes PabPol1His, an in-frame fusion of E. coli MBP with the seven C-terminal residues of the P. abyssi Pol II N-extein and the first 179 residues of the intein, fused to the C-terminal sequence K-F-Q-N-S-I-D-E-D-H-H-H-H-H-H. pPabPol1His was constructed by digesting pPabPol1 with EcoRI and HindIII and inserting the oligonucleotide pair PABUH (5'-AATTCTATCGATGAAGACCACCACCACCACCACCACTGA) and PABLH (5'-AGCTTCAGTGGTGGTGGTGGTGGTGGTCTTCATCGATAG).

Plasmid pPolWT was constructed to complete the intein sequence. pPolWT encodes PolWT, an in-frame fusion of E. coli MBP with the seven C-terminal residues of the P. abyssi Pol II N-extein and the 185 residues of the intein, fused to the C-terminal sequence C-D-G-D-E-D-H-H-H-H-H-H. It was constructed by digesting pPabPol1His with XmnI and ClaI and ligating it with the oligonucleotide pair PabUWT (5'-TATTGTAACGCATCAATGTGATGG) and PabLWT (5'-CGCCATCACATTGATGCGTTACAATA).

To generate mutations of pPolWT, plasmid pPabPol1His was digested with XmnI and ClaI and ligated with appropriate oligonucleotides as above to generate pPolQN, pPolHA, pPolQNHA, and pPolCA, which encode PolQN, PolHA, PolQNHA, and PolCA, respectively (Fig. 2B).

View larger version (43K): [in this window] [in a new window]   FIG. 2. Analysis of fusion proteins. A, purification of fusion proteins. Fusion proteins were isolated as described under "Experimental Procedures." Approximately 0.7 µg of each protein was subjected to SDS-PAGE as described and stained with Coomassie Blue. B, amino acid sequence at C-terminal splice junction. Amino acid sequence of PolWT is consistent with the native intein sequence. Other proteins have identical amino acid sequences to PolWT except for mutations at C-terminal splice junction indicated in the sequence alignment.

Protein Expression and Purification—Plasmid-encoded proteins were overexpressed in E. coli BL21DE3 (Novagen, Madison, WI). The cultures were grown at 37 °C with shaking to a culture density (A₆₀₀) of about 0.7 and induced with isopropyl-1-thio--D-galactopyranoside (final concentration, 0.4 mM) at 22 °C overnight. The cells were harvested by centrifugation and resuspended in Buffer A (20 mM Bis-Tris Propane, pH 7.5, 250 mM NaCl) supplemented with 5 mM MgCl₂, 12 units/ml benzonase nuclease (Novagen), 100 µM phenylmethylsulfonyl fluoride, 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride HCl, 0.8 µM aprotinin, 0.05 mM bestatin, 0.015 mM E-64, 0.02 mM leupeptin hemisulfate, and 0.01 mM pepstatin A (Calbiochem, La Jolla, CA).

The cell resuspensions were disrupted by passage through a French Pressure cell and centrifuged at 15,000 x g at 4 °C for 30 min. The resulting lysates were each added to 1 ml of Talon metal affinity resin (BD Clontech, Palo Alto, CA), pre-equilibrated with Buffer A, and incubated for 30 min at room temperature with shaking. The resin was collected by centrifugation and washed batch-wise with 2 x 10 ml of Buffer A. The resin was transferred to a gravity flow column and washed with 10 column volumes of Buffer A and 10 column volumes of Buffer A supplemented with 10 mM imidazole. The proteins were eluted in 500-µl fractions with Buffer A supplemented with 100 mM imidazole (Buffer AI). The concentration of protein in each fraction was estimated by the Bradford method (17).

Protein Splicing Conditions—Except where indicated, the proteins in Buffer AI were supplemented with 2.0 mM Tris(2-carboxyethyl)phosphine, 10 mM EDTA, and 136 mM Bis-Tris Propane, pH 7.5 (which resulted in a final pH value of 7.8), and reactions were initiated by incubation at indicated temperatures. Splicing and/or cleavage reactions were terminated by the addition of New England Biolabs 3x SDS-PAGE buffer supplemented with DL-1,4-dithiothreitol. For determination of the pH dependence of protein reactions, the proteins were also supplemented with 136 mM Bis-Tris Propane at pH 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. The pH values of the final protein mixtures were determined experimentally and indicated below. All of the pH values were as measured at 23 °C.

Protein Analysis—The proteins were analyzed by SDS-PAGE, Western blot, and high resolution mass spectrometry. For SDS-PAGE, precast 4–20% gradient, Tris-Glycine iGels (Gradipore, Frenchs Forest, Australia) were used via the Laemmli method (18). The samples were mixed with 3x SDS Sample buffer (New England Biolabs) supplemented with 40 µM DL-1,4-dithiothreitol and boiled for 3 min before loading, along with broad range and prestained broad range protein markers (New England Biolabs). The gels were stained with Coomassie Brilliant Blue R-250.

For Western blots, the gels were blotted onto nitrocellulose at 100 V for 1 h. The blots were incubated for 30 min in 1% bovine serum albumin in PBS and then with 2 µg/ml His tag monoclonal antibody (Novagen) in PBS for 1 h, washed with 50 ml of PBS three times for 20 min, incubated with 1:10,000 dilution of goat anti-mouse IgG alkaline phosphatase conjugate (Novagen), and then washed again with 50 ml of PBS three times for 20 min. The blots were developed for about 5 min with Western Blue stabilized substrate for alkaline phosphatase (Promega, Madison, WI). The blots were washed with deionized water, incubated with 0.18 M trichloroacetic acid for 10 min, then washed, and stored in deionized water.

The gels were imaged using a VersaDoc 3000 (Bio-Rad) and analyzed by densitometry with Quantity One software (Bio-Rad) or National Institutes of Health ImageJ software. The percentage of protein splicing was calculated from densitometry data and is described by [(MH)/(MH + MIH)] x 100, in which MH is the spliced product, and MIH is the unspliced precursor protein. The percentage of N-terminal cleavage is described by [M/(M + MIH)] x 100, in which M is the N-terminal product of the N-terminal cleavage reaction. C-terminal cleavage is described by [(I)/(I + IH)] x 100, in which I is the product of C-terminal cleavage of IH, which is the C-terminal product of the N-terminal cleavage reaction.

Rate constants of splicing assume a first order rate expression and were calculated from the slopes of plots of ln (1 – [(MH)/(MH + MIH)]) versus time in seconds. The rate constants of N-terminal cleavage assume a pseudo-first order rate constant and were calculated from the slopes of plots of ln (1 – [(M)/(M + MIH)]) versus time in seconds.

High resolution mass spectrometry data were obtained at the University of Massachusetts Proteomics and Mass Spectrometry Facility (Worcester, MA). PolWT and PolQN were subjected to protein splicing conditions described above for 12 h at 50 °C. The samples were analyzed using a Finnigan LCQ ion trap mass spectrometer and samples were separated by reverse phase chromatography using a 300 Å, 1 x 15-mm C18p3 column (LCPackings, Sunnyvale, CA) with a 10-min linear gradient of 2–90% acetonitrile in 0.1% formic acid at 50 µl/min.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Purification of Fusion Proteins—We were able to isolate proteins consisting of a fusion of N-terminal E. coli MBP to the seven C-terminal N-extein residues, 185 intein residues, and six N-terminal C-extein residues of the P. abyssi PolII intein, fused to a C-terminal His tag. Upon purification at room temperature using immobilized metal affinity chromatography and analysis by SDS-PAGE, single bands corresponding to the expected molecular mass of 66.7 kDa were observed (Fig. 2A). It was also shown via Western blot using anti-His tag antibodies that these bands contained the C-terminal His tag (Figs. 3B and 4B). The amino acid substitutions in PolQN, PolCA, PolHA, and PolQNHA are given in Fig. 2B.

View larger version (28K): [in this window] [in a new window]   FIG. 3. Analysis of protein splicing of PolWT and PolQN. PolWT (0.95 µM) or PolQN (1.0 µM) were incubated under the splicing conditions described under "Experimental Procedures" for 18 h at 70 °C. A, SDS-PAGE analysis of protein splicing. Lanes 1 and 3 consist of purified 1.9 µg of PolWT and 2.0 µg of PolQN, respectively. Lanes 2 and 4 consist of 30 µl of protein splicing mixture. SDS-PAGE was performed as described under "Experimental Procedures." B, Western blot analysis of protein splicing. Identical SDS-PAGE analysis was run as in A, except that proteins were transferred to nitrocellulose and analyzed by Western blot as described under "Experimental Procedures" using anti-His tag antibody, which selectively identifies the unspliced precursor, MIH, and the spliced product, MH, but not the excised intein, I.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Analysis of N-terminal cleavage of PolHA and PolQNHA. PolHA (1.0 µM) and PolQNHA (0.95 µM) were incubated under the splicing conditions described under "Experimental Procedures" for 18hat70 °C. A, SDS-PAGE analysis of N-terminal cleavage. Lanes 1 and 3 consist of purified 2.0 µg of PolHA and 1.9 µg of PolQNHA, respectively. Lanes 2 and 4 consist of 30 µl of protein splicing mixture. SDS-PAGE performed as described under "Experimental Procedures." B, Western blot analysis of N-terminal cleavage. Identical SDS-PAGE analysis was run as in A, except that proteins were transferred to nitrocellulose and analyzed by Western blot as described under "Experimental Procedures" using anti-His tag antibody, which selectively identifies the unspliced precursor, MIH, and the resulting intein fragment from N-terminal cleavage, IH, but not the N-terminal cleavage product M or the product of intein C-terminal cleavage, I.

SDS-PAGE analysis of the 18 h, 70 °C incubation of PolWT and PolQN in the presence of 2 mM Tris(2-carboxyethyl)phosphine and 10 mM EDTA resulted in bands consistent with the formation of protein splicing products MH and I as well as residual MIH (Fig. 3A). The identity of these bands was verified by Western blot analysis with anti-His tag antibodies (Fig. 3B), because the bands corresponding to MIH and MH reacted with the antibody, and the band corresponding to the excised intein, I, did not.

Incubation of PolHA and PolQNHA at 70 °C for 18 h did not result in detectable protein splicing. Instead, SDS-PAGE analysis detected bands consistent with the production of M, I, and IH, in addition to residual MIH (Fig. 4A). Western blot analysis with anti-His tag antibodies showed that the bands corresponding to MIH and IH had the His tag, and bands corresponding to M and I did not (Fig. 4B).

The presence of the excised intein for PolHA and PolQNHA suggests that either the cleaved IH band is capable of C-terminal cleavage after N-terminal cleavage, that C-terminal cleavage of MIH precedes N-terminal cleavage, or a mixture of both. It is difficult to distinguish MIH (66.7 kDa) from MI (65.3 kDa) via SDS-PAGE because of the small size of the C-extein used in the fusion protein.

Mass Spectrometry of Excised Inteins—To determine whether we could detect any modification of the C-terminal intein residue during protein splicing, PolWT and PolQN were incubated under splicing conditions at 50 °C for 12 h and analyzed via high resolution ion trap liquid chromatography-mass spectrometry. A peak with a deconvoluted mass of 21521.0 Da was detected in the PolWT splicing mixture, consistent with the predicted molecular mass of 21522.3 Da. For splicing with PolQN, a peak with a deconvoluted mass of 21509.0 Da was detected, consistent with the predicted value of 21508.3 Da.

Temperature Dependence of Protein Splicing and Cleavage Reactions—To study the effect of temperature on protein splicing, PolWT and PolQN were incubated under splicing conditions for 18 h at different temperatures. No detectable protein splicing or cleavage reactions were observed after 18 h at 4 °C or 20 °C for either protein. Protein splicing increased with temperature for both constructs at 30 °C and above, with PolQN reaching a plateau of 80–85% splicing between 50 and 70 °C (Fig. 5A). Protein splicing of the wild-type construct PolWT reached 74% at 70 °C (Fig. 5A).

View larger version (13K): [in this window] [in a new window]   FIG. 5. Temperature dependence of protein splicing and N- and C-terminal cleavage reactions. A, the effect of temperature on splicing of PolQN () and PolWT () was examined by incubation of 1.3 µM PolWT or 1.0 µM PolQN under the protein splicing conditions described under "Experimental Procedures" for 18 h at 60 °C, except that Bis-Tris Propane of pH 6.5 was used for a final pH of the mixture of 7.1. The reactions were analyzed by SDS-PAGE and analyzed as described under "Experimental Procedures." B, the effect of temperature on N-terminal cleavage of PolQNHA (), PolHA (), and PolCA () was examined by incubation of 1.4 µM PolCA, 1.3 µM PolHA, or 1.7 µM PolQNHA as described for the protein splicing conditions in A. C, the effect of temperature on C-terminal cleavage of the IH fragment was examined for 1.7 µM PolQNHA under the same conditions described in A.

C-terminal cleavage was detected for PolHA, PolQNHA, and PolCA. For PolQNHA, this cleavage was plotted as a function of temperature. C-terminal cleavage was detectable for PolQNHA only at 50 °C and above, higher than for N-terminal cleavage of this construct (Fig. 5C). We have defined C-terminal cleavage as the ratio of band I to bands I plus IH, assuming that all C-terminal cleavage is of the N-terminal cleavage product.

Rate Constants for Splicing and N-terminal Cleavage Reactions—We have determined the rate constants for protein splicing and N-terminal cleavage (Tables I and II). The rate constant for protein splicing for PolQN is from 2.8 to 3.6 times that of PolWT at all temperatures tested.

Dependence of Splicing and Cleavage Reactions on pH—For PolWT and PolQN, protein splicing was most optimal between pH values of 6.8 and 7.4 and declined at higher pH values (Fig. 6A). However, N-terminal cleavage of PolHA and PolQNHA was optimal between pH 7.4 and 7.8 (Fig. 6B). The pH profile of N-terminal cleavage of PolCA was more consistent with that of the protein splicing of PolWT and PolQN than the N-terminal cleavage of PolHA and PolQNHA (Fig. 6).

View larger version (15K): [in this window] [in a new window]   FIG. 6. Protein splicing and N-terminal cleavage dependence on pH of reaction mixture. A, the effect of pH on splicing of PolQN () and PolWT () was examined by incubation of 1.3 µM PolWT or 1.0 µM PolQN under the protein splicing conditions described under "Experimental Procedures" for 18 h at 60 °C, except that Bis-Tris Propane of pH 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 was used. The final pH value for the reaction mixtures were determined experimentally and used for data plotting. The reactions were analyzed by SDS-PAGE and analyzed as described under "Experimental Procedures." B, the effect of pH on N-terminal cleavage of PolQNHA (), PolHA (), and PolCA () was examined by incubation of 1.4 µM PolCA, 1.3 µM PolHA, or 1.7 µM PolQNHA as described for the protein splicing conditions in A.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The PolII intein facilitates protein splicing both with a C-terminal Gln as well as with a C-terminal Gln-Asn substitution. Although the substitution of Asn for Gln in the CIV RNR intein resulted in a 7-fold reduction in protein splicing yield (10), both the yield and rate of protein splicing were moderately improved by substitution of Asn for Gln in the PolII intein (Fig. 3 and Tables I and II). The similarity in behavior of PolWT and PolQN suggests that deprotonation of the side chain is unlikely to play a significant role in the catalysis of protein splicing for the wild-type P. abyssi PolII intein.

Mutation of the penultimate His residue of the P. abyssi PolII intein ablated splicing activity and led to temperature-dependent N-terminal cleavage (Figs. 4 and 5B). However, the PolHA and PolQNHA mutant inteins are capable of promoting C-terminal cleavage (Figs. 4 and 5C). In addition, C-terminal cleavage of PolQNHA is initiated at higher temperatures than N-terminal cleavage of PolQNHA or splicing of PolQN (Fig. 5). This suggests a role for the penultimate His in coordinating the rate of each of the steps of the protein splicing reaction (Fig. 1). It is unclear whether N-terminal cleavage must precede C-terminal cleavage, as is the case with a subset of other reported inteins (20, 21).

Mutation of the first Cys residue of the C-extein also results in in vitro N-terminal cleavage (Figs. 5B and 6B). The fact that N-terminal cleavage still occurs suggests that hydrolysis of the linear thioester intermediate is responsible. However, the increased yield and rate of N-terminal cleavage of the PolHA and PolQNHA mutants with respect to PolCA (Figs. 5 and 6 and Tables I and II) suggests that hydrolysis of the branched thioester intermediate may contribute to N-terminal cleavage of PolHA and PolQNHA. This would be consistent with observations involving the Synechocystis sp. PCC6803 DnaE intein, in which modulation of the +1 Cys residue by mutation or binding of ZnCl₂ inhibited spontaneous N-terminal cleavage, most likely by blocking transesterification (22).

The third step of protein splicing for inteins with a C-terminal asparagine residue has been proposed to be the cyclization of asparagine coupled to peptide bond cleavage (1). This hypothesis has been supported by the isolation and detection of excised inteins with C-terminal succinimide residues (2, 3, 4). Upon incubation of PolWT and PolQNHA under splicing conditions at 50 °C for 12 h, we detected the excised intein by high resolution mass spectrometry. However, we could not detect a peak with a mass of 18 Da less than expected for the excised intein, which would have been consistent with the loss of water associated with a cyclized C-terminal Gln for PolWT or a cyclized C-terminal Asn for PolQN. Attempts to detect a C-terminal succinimide residue for the Q339N CIV RNR intein were also unsuccessful, as were attempts to detect a C-terminal glutarimide in the wild-type CIV RNR intein (10). It is not surprising that we were unable to detect a cyclized C-terminal glutarimide residue, because glutarimides have been shown to be 100 times more reactive to hydrolysis than succinimides (23–25). However, the failure to detect a C-terminal succinimide residue for PolQN suggests that inteins with C-terminal Gln may splice via a mechanism other than side chain cyclization, even upon substitution of Gln with Asn.

The failure to detect a succinimide residue at the C terminus of the excised Gln-Asn mutant of the CIV RNR intein led the authors of that study to suggest an alternate mechanism for the resolution of the branched intermediate in protein splicing (10). Instead of cyclization of the C-terminal glutamine by nucleophilic attack of the side chain amide nitrogen (coupled to peptide bond cleavage) to form a glutarimide (Fig. 1, step C), they proposed that the side chain carbonyl oxygen serves as the nucleophile to couple peptide bond cleavage to formation of a C-terminal glutaranhydride (Fig. 1, step D). The formation of a less stable anhydride intermediate might account for the inability to detect a C-terminal cyclized residue, especially in the C-terminal Gln-Asn mutants. Although there is no experimental evidence to support this proposal, it is attractive because one would expect the carbonyl oxygen to be a better nucleophile than the amide nitrogen. Also, the C. hydrogenoformus RNR intein is capable of facilitating protein splicing with a C-terminal Asp, which could also splice via a succinanhydride intermediate (10). In addition, the C-terminal cleavage of a C. hydrogenoformus RNR intein in which the C-terminal Asp is mutated to Ala suggests that an alternate mechanism for C-terminal cleavage that does not involve amino acid cyclization might exist (10). One such mechanism was suggested by the authors of the CIV RNR study (10) and originally proposed by A.L. Nussbaum (26). They proposed that the upstream N-terminal Cys residue of the intein could serve as the nucleophile that attacks the C-terminal scissile bond, resulting in peptide bond cleavage coupled to intein cyclization (Fig. 1, step E). Although there is no experimental evidence that supports this mechanism, it is one possible explanation for the inability to isolate an intein with a cyclized C-terminal amino acid for inteins with C-terminal Gln or Asp.

Because we were able to study the reactivity of the P. abyssi PolII intein in vitro, it was possible to determine rate constant data for the splicing and N-terminal cleavage reactions. However, incubation of the intein fusion proteins in vitro at the 95 °C growth temperature of P. abyssi resulted in protein precipitation, as did incubation at temperatures above 75 °C (data not shown), so we were unable to calculate rate constants at the temperature the intein would experience in vivo. Interestingly, attempts to determine whether the intein was inhibited by the presence of zinc ion, as observed with other inteins (22, 28, 29), were unsuccessful because incubation of each of the PolII intein fusion proteins with zinc chloride also resulted in protein precipitation (data not shown).

The rates of protein splicing and N-terminal cleavage of the P. abyssi PolII intein constructs are very similar. For PolHA, the pseudo-first order rate constant of N-terminal cleavage at 65 °C was determined to be 9.8 x 10^–6 s^–1, as opposed to the calculated first order rate constant of splicing for PolWT at 65 °C of 1.3 x 10^–5 s^–1. Likewise, the pseudo-first order rate constant of N-terminal cleavage for PolQNHA at 65 °C was 1.8 x 10^–5 s^–1, whereas that for splicing of PolQN was 4.7 x 10^–5 s^–1. The fact that the splicing rates are slightly higher than the cleavage rates is somewhat unexpected but could be explained by the mutation altering the conformation of the His-Ala mutant inteins. The observation that N-terminal cleavage occurs at a similar rate to splicing could also indicate that either transesterification or the initial N-S acyl rearrangement is the rate-determining step for splicing of the P. abyssi PolII intein. In the most thorough kinetic characterization of an intein, that of the Synechocystis sp. PCC6803 DnaE intein, the rate of N-terminal cleavage was about 15 times higher than the rate for splicing (21). However, in this system N-terminal cleavage was of the linear thioester because the N-terminal Cys of the C-extein was not present (21), so it is possible that transesterification was the slow step of splicing for the Synechocystis sp. PCC6803 DnaE intein. Although the reported first order rate constants of protein splicing for the Synechocystis sp. PCC6803 DnaE intein range from 6.6 x 10^–5 s^–1 at 23 °C (21) to 3.3 x 10^–4 s^–1 at room temperature (22), splicing of the P. abyssi PolII intein proceeded with a first order rate constant of 3.9 x 10^–6 s^–1 for PolWT and 1.1 x 10^–5 s^–1 for PolQN at 40 °C (Tables I and II). However, a more apt comparison of protein splicing rates might be to compare rates at physiological temperatures. Although it was not possible to measure protein splicing rates of the P. abyssi PolII intein in vitro at 95 °C, an extrapolation of the rate data for splicing using the Arrhenius equation would give a first order rate constant at 95 °Cof3.8 x 10^–5 s^–1 for PolWT and 1.8 x 10^–4 s^–1 for PolQN, which are in better agreement with the Synechocystis sp. PCC6803 DnaE intein data. The first order rate of DL-1,4-dithiothreitol -induced N-terminal cleavage of the Synechocystis sp. PCC6803 DnaE intein was reported as 1.0 x 10^–3 s^–1 at 23 °C (21), whereas the pseudo-first order rate constant of N-terminal cleavage of PolHA is 9.8 x 10^–6 s^–1 at 65 °C (Tables I and II). Although we do not have sufficient data to estimate an N-terminal cleavage rate at 95 °C for the P. abyssi PolII intein, it is interesting to note that for the Synechocystis sp. PCC6803 DnaE intein, the rate constant of N-terminal cleavage is about 15 times that for splicing (21), whereas the rate constants of N-terminal cleavage and splicing of the P. abyssi PolII inteins are about the same.

Protein splicing of PolWT and PolQN is dependent on the pH of the reaction mixture. Splicing was optimal between pH 6.8 and 7.4 and declined at higher pH values (Fig. 6). This is consistent with data observed for other inteins (21, 30, 31). N-terminal cleavage did not decline as much at high pH as did splicing, again consistent with previous data (21) and possibly because of competition between alkaline hydrolysis and reversion to the amide-linked precursor.

The characterization of the P. abyssi PolII intein, an intein that splices via a noncanonical mechanism, adds to our understanding of the diversity of intein biochemistry, which includes the recent discovery of the bacterial intein-like protein domains (32). It should also be possible to use the P. abyssi PolII intein todevelopaproteinpurificationsystembasedonthetemperature-dependent N-terminal cleavage properties of the fusion proteins with penultimate His-Ala mutations. For instance, N-terminal cleavage of PolQNHA reached 40% at 50 °C after 18 h, whereas C-terminal cleavage was about 5% (Fig. 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).

	FOOTNOTES

 
^* This work was supported by the Donors of the Petroleum Research Fund, administered by the American Chemical Society. This work was also supported by the Research Corporation via a Cottrell College Science Award, National Science Foundation MRI Grant DBI-0320824, and summer salary support from the Simeon J. Fortin Charitable Trust (to J. S. M., A. M. G., and L. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number E75199 [GenBank] .

To whom correspondence should be addressed: College of the Holy Cross, Dept. of Chemistry, 1 College St., Worcester, MA 01610. Tel.: 508-793-3380; Fax: 508-793-3530; E-mail: kmills{at}holycross.edu.

¹ The abbreviations used are: C-extein, C-terminal extein; RNR, ribonucleotide reductase; Bis-Tris Propane, 1,3-bis-[tris(hydroxymethyl)-methylamine] propane; CIV, Chilo iridescent virus; MBP, E. coli maltose-binding protein; H, the six N-terminal residues of the P. abyssi PolII C-extein fused to a C-terminal His tag; I, the Pab PolII intein; M, N-terminal extein containing N-terminal MBP, a spacer, and the seven C-terminal residues of the Pab PolII N-extein; N-extein, N-terminal extein; PBS, phosphate-buffered saline; PolII, DNA polymerase II. The nomenclature of P. abyssi PolII intein mutants is listed schematically in Fig 2B.

	ACKNOWLEDGMENTS

 
We thank the Holy Cross Department of Biology for the use of facilities. We acknowledge Prof. Frank Robb (Center for Marine Biotechnology, University of Maryland Biotechnology Institute) for providing a culture of P. abyssi and Prof. Raymond Cunin (Laboratorium van Microbiologie, Vrije Universiteit Brussel) for providing a sample of P. abyssi genomic DNA. We also gratefully acknowledge Prof. Madeline Vargas for expertise in the growth of thermophilic bacteria and use of facilities and Profs. Robert Bellin and Kevin Quinn for useful discussions and a critical reading of the manuscript.

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