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Tom W Muir - One of the best experts on this subject based on the ideXlab platform.
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an atypical mechanism of split intein molecular recognition and folding
2018Co-Authors: Adam J. Stevens, Josef A. Gramespacher, David Cowburn, Giridhar Sekar, Tom W MuirAbstract:Split inteins associate to trigger Protein Splicing in trans, a post-translational modification in which Protein sequences fused to the intein pair are ligated together in a traceless manner. Recently, a family of naturally split inteins has been identified that is split at a noncanonical location in the primary sequence. These atypically split inteins show considerable promise in Protein engineering applications; however, the mechanism by which they associate is unclear and must be different from that of previously characterized canonically split inteins due to unique topological restrictions. Here, we use a consensus design strategy to generate an atypical split intein pair (Cat) that has greatly improved activity and is amenable to detailed biochemical and biophysical analysis. Guided by the solution structure of Cat, we show that the association of the fragments involves a disorder-to-order structural transition driven by hydrophobic interactions. This molecular recognition mechanism satisfies the topological constraints of the intein fold and, importantly, ensures that premature chemistry does not occur prior to fragment complementation. Our data lead a common blueprint for split intein complementation in which localized structural rearrangements are used to drive folding and regulate Protein-Splicing activity.
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Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-Splicing
2016Co-Authors: Neel H. Shah, David Cowburn, Ertan Eryilmaz, Tom W MuirAbstract:ABSTRACT: Split inteins play an important role in modern Protein semisynthesis techniques. These naturally occurring Protein Splicing domains can be used for in vitro and in vivo Protein modification, peptide and Protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice Proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N- and C-exteins) are strongly preferred, while other sequences cause a dramatic reduction in the Splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in Protein Splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation tha
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design of a split intein with exceptional Protein Splicing activity
2016Co-Authors: Adam J. Stevens, David Cowburn, Neel H. Shah, Giridhar Sekar, Zachary Z Brown, Tom W MuirAbstract:Protein trans-Splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in Splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the Protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid Protein Splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various Proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The du...
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Design of a Split Intein with Exceptional Protein Splicing Activity
2016Co-Authors: Adam J. Stevens, David Cowburn, Neel H. Shah, Zachary Z. Brown, Giridhar Sekar, Tom W MuirAbstract:Protein trans-Splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in Splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the Protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid Protein Splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various Proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new Protein chemistry techniques
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Ultrafast Protein Splicing is Common among Cyanobacterial Split Inteins: Implications for Protein Engineering
2016Co-Authors: Neel H. Shah, Geoffrey P. Dann, Miquel Vila-perelló, Zhihua Liu, Tom W MuirAbstract:We describe the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria. By measuring in vivo Splicing efficiencies and in vitro kinetics, we demonstrate that several inteins can catalyze Protein trans-Splicing in tens of seconds rather than hours, as is commonly observed for this autoprocessing Protein family. Furthermore, we show that when artificially fused, these inteins can be used for rapid generation of Protein α-thioesters for expressed Protein ligation. This comprehensive survey of split inteins provides indispensable information for the development and improvement of intein-based tools for chemical biology
Kenneth V. Mills - One of the best experts on this subject based on the ideXlab platform.
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Protein Splicing activity of the haloferax volcanii polb c intein is sensitive to homing endonuclease domain mutations
2020Co-Authors: Shachar Robinzon, Uri Gophna, Alexandra R Cawood, Mercedes Ruiz, Neta Altmanprice, Kenneth V. MillsAbstract:Inteins are selfish genetic elements residing in open reading frames that can splice post-translationally, resulting in the ligation of an uninterrupted, functional Protein. Like other inteins, the DNA polymerase B (PolB) intein of the halophilic archaeon Haloferax volcanii has an active homing endonuclease (HEN) domain, facilitating its horizontal transmission. Previous work has shown that the presence of the PolB intein exerts a significant fitness cost on the organism compared to an intein-free isogenic H. volcanii. Here, we show that mutation of a conserved residue in the HEN domain not only reduces intein homing but also slows growth. Surprisingly, although this mutation is far from the Protein Splicing active site, it also significantly reduces in vitro Protein Splicing. Moreover, two additional HEN domain mutations, which could not be introduced to H. volcanii, presumably due to lethality, also eliminate Protein Splicing activity in vitro. These results suggest an interplay between HEN residues and the Protein Splicing domain, despite an over 35 A separation in a PolB intein homology model. The combination of in vivo and in vitro evidence strongly supports a model of codependence between the self-Splicing domain and the HEN domain that has been alluded to by previous in vitro studies of Protein Splicing with HEN domain-containing inteins.
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Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N‑Terminal Cleavage
2017Co-Authors: Christopher J. Minteer, Julie N. Reitter, Robert J. Linhardt, Kathryn M. Colelli, Nicolle M. Siegart, Xinyue Liu, Alvin V. Gomez, Kenneth V. MillsAbstract:Protein Splicing is a post-translational reaction facilitated by an intein, or intervening Protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote Protein Splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of Splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar “aspartic acid effects” have been observed in other Proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of Protein Splicing and provide insight into the stability of Proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology
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salt dependent conditional Protein Splicing of an intein from halobacterium salinarum
2016Co-Authors: Julie N. Reitter, Michael Nicastri, Christopher E Cousin, Mario Jaramillo, Kenneth V. MillsAbstract:An intein from Halobacterium salinarum can be isolated as an unspliced precursor Protein with exogenous exteins after Escherichia coli overexpression. The intein promotes Protein Splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The Protein Splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional Protein Splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.
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Internal Disulfide Bond Acts as a Switch for Intein Activity
2015Co-Authors: Michael Nicastri, Kristina Xega, Julie N. Reitter, Robert J. Linhardt, Jian Xie, Kenneth V. MillsAbstract:ABSTRACT: Inteins are intervening polypeptides that catalyze their own removal from flanking exteins, concomitant to the ligation of the exteins. The intein that interrupts the DP2 (large) subunit of DNA polymerase II from Methanoculleus marisnigri (Mma) can promote Protein Splicing. However, Protein Splicing can be prevented or reduced by overexpression under nonreducing conditions because of the formation of a disulfide bond between two internal intein Cys residues. This redox sensitivity leads to differential activity in different strains of E. coli as well as in different cell compartments. The redox-dependent control of in vivo Protein Splicing in an intein derived from an anaerobe that can occupy multiple environments hints at a possible physiological role for Protein Splicing. Protein Splicing is a self-catalyzed process facilitated by anintein. The intein interrupts two flanking polypeptides, called the N- and C-exteins, and promotes both its own excision and the ligation of these exteins.1,2 Protein Splicing usually follows a four-step mechanism (Figure 1A). First, the peptide bond linking the N-extein an
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Protein Splicing: how inteins escape from precursor Proteins.
2014Co-Authors: Kenneth V. Mills, Margaret A. Johnson, Francine B. PerlerAbstract:Inteins are nature's escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host Protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the Splicing steps are controlled.
Francine B. Perler - One of the best experts on this subject based on the ideXlab platform.
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Branched Intermediate Formation Is the Slowest Step in the Protein Splicing Reaction of the Ala1 KlbA Intein from Methanococcus jannaschii
2015Co-Authors: Lana Saleh, Jack Benner, Maurice W. Southworth, Nancy Considine, Colleen O’neill, Martin J. Bollinger, Francine B. PerlerAbstract:We report the first detailed investigation of the kinetics of Protein Splicing by the Methanococcus jannaschii KlbA (Mja KlbA) intein. This intein has an N-terminal Ala in place of the nucleophilic Cys or Ser residue that normally initiates Splicing but nevertheless splices efficiently in vivo [Southworth, M. W., Benner, J., and Perler, F. B. (2000) EMBO J. 19, 5019–5026]. To date, the spontaneous nature of the cis Splicing reaction has hindered its examination in vitro. For this reason, we constructed an Mja KlbA intein–mini-extein precursor using intein-mediated Protein ligation and engineered a disulfide redox switch that permits initiation of the Splicing reaction by the addition of a reducing agent such as dithiothreitol (DTT). A fluorescent tag at the C-terminus of the C-extein permits monitoring of the progress of the reaction. Kinetic analysis of the Splicing reaction of the wild-type precursor (with no substitutions in known nucleophiles or assisting groups) at various DTT concentrations shows that formation of the branched intermediate from the precursor is reversible (forward rate constant of 1.5 × 10–3 s–1 and reverse rate constant of 1.7 × 10–5 s–1 at 42 °C), whereas the productive decay of this intermediate to form the ligated exteins is faster and occurs with a rate constant of 2.2 × 10–3 s–1. This finding conflicts with reports about standard inteins, for which Asn cyclization has been assigned as the rate-determining step of the Splicing reaction. Despite being the slowest step of the reaction, branched intermediate formation in the Mja KlbA intein is efficient in comparison with those of other intein systems. Interestingly, it also appears that this intermediate is protected against thiolysis by DTT, in contrast to other inteins. Evidence is presented in support of a tight coupling between the N-terminal and C-terminal cleavage steps, despite the fact that the C-terminal single-cleavage reaction occurs in variant Mja KlbA inteins in the absence of N-terminal cleavage. We posit that the Splicing events in the Mja KlbA system are tightly coordinated by a network of intra- and interdomain noncovalent interactions, rendering its function particularly sensitive to minor disruptions in the intein or extein environments
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Protein Splicing: how inteins escape from precursor Proteins.
2014Co-Authors: Kenneth V. Mills, Margaret A. Johnson, Francine B. PerlerAbstract:Inteins are nature's escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host Protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the Splicing steps are controlled.
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faster Protein Splicing with the nostoc punctiforme dnae intein using non native extein residues
2013Co-Authors: Manoj Cheriyan, Chandra Sekhar Pedamallu, Kazuo Tori, Francine B. PerlerAbstract:Inteins are naturally occurring intervening sequences that catalyze a Protein Splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing Proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon Splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect Splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for Splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote Splicing in three unrelated Proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed Splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for Splicing.
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Protein Splicing in cis and in trans
2006Co-Authors: Lana Saleh, Francine B. PerlerAbstract:Intein-mediated Protein Splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor Protein and the flanking extein segments are ligated with a native peptide bond. Splice junction proximal residues and internal residues within the intein direct these reactions. The identity of these residues varies in each intein, as groups of related residues populate conserved motifs. Although the basics of the four-step Protein Splicing pathway are known, mechanistic details are still unknown. Structural and kinetic analyses are beginning to shed some light. Several structures were reported for precursor Proteins with mutations in catalytic residues, which stabilize the precursors for crystallographic study. Progress is being made despite limitations inherent in using mutated precursors. However, no uniform mechanism has emerged. Kinetic parameters were determined using conditional trans-Splicing (Splicing of split precursor fragments after intein reassembly). Several groups concluded that the rate of the initial acyl rearrangement step is rapid and Asn cyclization (step 3) is slow, suggesting that this latter step is rate limiting. Understanding the Protein Splicing pathway has allowed scientists to harness inteins for numerous applications. © 2006 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 6: 183–193; 2006: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20082
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An alternative Protein Splicing mechanism for inteins lacking an N-terminal nucleophile
2000Co-Authors: Maurice W. Southworth, Jack S. Benner, Francine B. PerlerAbstract:Variations in the intein-mediated Protein Splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard Protein Splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative Protein Splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.
Henry Paulus - One of the best experts on this subject based on the ideXlab platform.
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stilbenoids from hopea acuminata
2016Co-Authors: Christine Chichioco L Hernandez, Henry Paulus, Irene M Villasenor, Frank C Schroeder, Jon ClardyAbstract:Two resveratrol oligomers, a dimer (1) and a tetramer (2), were isolated from the ethyl acetate extracts of the leaves of Hopea acuminata along with vaticanol B, vaticaphenol A, vateriaphenol B, hopeaphenol, e-viniferindiol, balanocarpol, and ampelopsin A. The structures of these compounds were established on the basis of spectroscopic data including two-dimentional nuclear magnetic resonance experiments. Compound 2, vaticanol B, vaticaphenol A, vateriaphenol B, hopeaphenol were found to inhibit Protein Splicing mediated by the Mycobacterium tuberculosis RecA intein in a nonspecific manner.
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An in vivo screening system against Protein Splicing useful for the isolation of non-Splicing mutants or inhibitors of the RecA intein of Mycobacterium tuberculosis.
2001Co-Authors: Belinda M Lew, Henry PaulusAbstract:Protein Splicing involves the self-catalyzed excision of an intervening sequence, the intein, from a precursor Protein, with the concomitant ligation of the flanking extein sequences to yield a new polypeptide. The ability of inteins to promote Protein Splicing even when inserted into a foreign context has facilitated the study of the modulation of Protein Splicing. In this paper, we describe an in vivo screening system for the isolation of mutations or inhibitors that interfere with Protein Splicing mediated by the RecA intein of Mycobacterium tuberculosis. It involves the activation of the cytotoxic CcdB Protein by Protein Splicing, such that host cells survive in the presence of inducer only when Protein Splicing is blocked. The coding sequence for the RecA intein was inserted in-frame into the polylinker region of an inducible lacZα-ccdB fusion vector, leading to inactivation of the CcdB toxin unless the intein is excised by Protein Splicing. Depending on the objective of the screening procedure, its stringency can be modified by altering the level of expression of the intein-CcdB fusion Protein. To induce large amounts of CcdB fusion Proteins, the fusion Protein is expressed from a high-copy-number plasmid. Such a screening system detects even low levels of Protein Splicing and we have used it to show that Protein Splicing of the RecA intein is compatible with any amino acid in the extein position adjacent to the N-terminal splice junction. In order to search for Protein Splicing inhibitors, which may attenuate Protein Splicing by less than an order of magnitude, we have also constructed a low-copy-number intein-CcdB plasmid so that the host cells can survive when Splicing of the expressed CcdB fusion Protein is only moderately suppressed. We anticipate that the CcdB-based in vivo screening system will find uses in the analysis of structural and mechanistic aspects of Protein Splicing.
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reactivity of the cysteine residues in the Protein Splicing active center of the mycobacterium tuberculosis reca intein
2000Co-Authors: Kaori Shingledecker, Henry Paulus, Shuqin JiangAbstract:Abstract Protein Splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor Protein. The first two steps in the Protein Splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion Proteins containing a truncated RecA intein modified by mutation to prevent Protein Splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent p K a values of the cysteines at the two splice junctions. The apparent p K a of the cysteine at the upstream splice junction, which initiates the N–S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low p K a of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.
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Protein Splicing and related forms of Protein autoprocessing.
2000Co-Authors: Henry PaulusAbstract:▪ Abstract Protein Splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a Protein, accompanied by the concomitant joining of the flanking polypeptide sequences, the exteins, by a peptide bond. It requires neither cofactors nor auxiliary enzymes and involves a series of four intramolecular reactions, the first three of which occur at a single catalytic center of the intein. Protein Splicing can be modulated by mutation and converted to highly specific self-cleavage and Protein ligation reactions that are useful Protein engineering tools. Some of the reactions characteristic of Protein Splicing also occur in other forms of Protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonProtein moieties. These mechanistic similarities may be the result of convergent evolution, but in at least one case—hedgehog Protein autoprocessing—there is definitely a close evolutionary relationship to Protein Splicing.
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Protein Splicing in trans by purified n and c terminal fragments of the mycobacterium tuberculosis reca intein
1998Co-Authors: Kenneth V. Mills, Shuqin Jiang, Henry PaulusAbstract:Abstract Protein Splicing involves the self-catalyzed excision of Protein Splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation of new Proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N- and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each ≈100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional Protein Splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted Splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo Protein Splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional Protein Splicing element and for the Protein Splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and Splicing most rapid at pH 7.0. The availability of such an in vitro Protein Splicing system opens the way for the exploration of intein structure and the unusual enzymology of Protein Splicing. In addition, this trans-Splicing system is a potential Protein ligase that can link any two polypeptides fused to the N- and C-terminal intein segments.
Marlene Belfort - One of the best experts on this subject based on the ideXlab platform.
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spliceosomal prp8 intein at the crossroads of Protein and rna Splicing
2019Co-Authors: Cathleen M Green, Nilesh K. Banavali, O S Novikova, Valjean R Bacotdavis, Aaron D Smith, Fengshan Gao, Dennis J Thiele, Marlene BelfortAbstract:The spliceosome is a large ribonucleoProtein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) Protein. Across diverse eukaryotes, this Protein cofactor of RNA catalysis harbors a self-Splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among Protein Splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein Splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 Protein Splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced Protein Splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 Protein Splicing can be modulated, with potential functional implications for the spliceosome.
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Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing.
2019Co-Authors: Christopher W. Lennon, Matthew J. Stanger, Marlene BelfortAbstract:Inteins, or intervening Proteins, are mobile genetic elements translated within host polypeptides and removed through Protein Splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host Protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional Protein Splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading Protein function. The RadA recombinase from the archaeon Pyrococcus horikoshii represents an intriguing example of CPS, whereby Protein Splicing is inhibited by interactions between the intein and host Protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the Splicing rate and accura...
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spliceosomal prp8 intein at the crossroads of Protein and rna Splicing
2018Co-Authors: Cathleen M Green, Marlene Belfort, Nilesh K. Banavali, O S Novikova, Valjean R Bacotdavis, Fenghan GaoAbstract:The spliceosome is a large ribonucleoProtein complex that removes introns from pre-mRNAs. At its functional core lies the essential Prp8 Protein. Across diverse eukaryotes, this Protein cofactor of RNA catalysis harbors a self-Splicing element, called an intein. Inteins in Prp8 are extremely pervasive and are found at seven different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans, a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among self-Splicing sequences in eukaryotes, including Hedgehog Protein. Working with the C. neoformans Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein Splicing, albeit by two different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds via the same critical cysteine with a Kd of ~1 nM. An intein-containing Prp8 precursor model is presented, suggesting that metal-induced Protein Splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 Protein Splicing can be modulated, and that this could alter spliceosome function and RNA Splicing under specific conditions.
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Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership
2018Co-Authors: Christopher W. Lennon, Marlene Belfort, Matthew J. Stanger, Nilesh K. Banavali, Jeff F. MillerAbstract:Inteins are intervening Proteins that undergo an autocatalytic Splicing reaction that ligates flanking host Protein sequences termed exteins. Some intein-containing Proteins have evolved to couple Splicing to environmental signals; this represents a new form of posttranslational regulation. Of particular interest is RadA from the archaeon Pyrococcus horikoshii, for which long-range intein-extein interactions block Splicing, requiring temperature and single-stranded DNA (ssDNA) substrate to splice rapidly and accurately. Here, we report that Splicing of the intein-containing RadA from another archaeon, Thermococcus sibericus, is activated by significantly lower temperatures than is P. horikoshii RadA, consistent with differences in their growth environments. Investigation into variations between T. sibericus and P. horikoshii RadA inteins led to the discovery that a nonconserved region (NCR) of the intein, a flexible loop where a homing endonuclease previously resided, is critical to Splicing. Deletion of the NCR leads to a substantial loss in the rate and accuracy of P. horikoshii RadA Splicing only within native exteins. The influence of the NCR deletion can be largely overcome by ssDNA, demonstrating that the Splicing-competent conformation can be achieved. We present a model whereby the NCR is a flexible hinge which acts as a switch by controlling distant intein-extein interactions that inhibit active site assembly. These results speak to the repurposing of the vestigial endonuclease loop to control an intein-extein partnership, which ultimately allows exquisite adaptation of Protein Splicing upon changes in the environment
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Protein Splicing of a recombinase intein induced by ssdna and dna damage
2016Co-Authors: Christopher W. Lennon, Matthew J. Stanger, Marlene BelfortAbstract:Inteins (or Protein introns) autocatalytically excise themselves through Protein Splicing. We challenge the long-considered notion that inteins are merely molecular parasites and posit that some inteins evolved to regulate host Protein function. Here we show substrate-induced and DNA damage-induced Splicing, in which an archaeal recombinase RadA intein splices dramatically faster and more accurately when provided with ssDNA. This unprecedented example of intein Splicing stimulation by the substrate of the invaded host Protein provides compelling support in favor of inteins acting as pause buttons to arrest Protein function until needed; then, an immediate activity switch is triggered, representing a new form of post-translational control.
