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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, Maurice W. Southworth, Nancy Considine, Colleen O’neill, Jack Benner, 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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Traceless splicing enabled by substrate-induced activation of the Nostoc punctiforme Npu DnaE intein after mutation of a catalytic cysteine to serine.
Journal of molecular biology, 2014Co-Authors: Manoj Cheriyan, Siu-hong Chan, Francine B. PerlerAbstract:Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding Extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-Extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified Extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-Extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices.
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Protein splicing: how inteins escape from precursor proteins.
The Journal of biological chemistry, 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
Journal of Biological Chemistry, 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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Branched intermediate formation is the slowest step in the protein splicing reaction of the Ala1 KlbA intein from Methanococcus jannaschii.
Biochemistry, 2011Co-Authors: Lana Saleh, Maurice W. Southworth, Jack S. Benner, Nancy Considine, Colleen O’neill, J. Martin 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 tha...
Kenneth V. Mills - One of the best experts on this subject based on the ideXlab platform.
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Methods to Study the Structure and Catalytic Activity of cis-Splicing Inteins.
Methods in molecular biology (Clifton N.J.), 2020Co-Authors: Jing Zhao, Chunyu Wang, Kenneth V. MillsAbstract:The autocatalytic process of protein splicing is facilitated by an intein, which interrupts flanking polypeptides called Exteins. The mechanism of protein splicing has been studied by overexpression in E. coli of intein fusion proteins with nonnative Exteins. Inteins can be used to generate reactive α-thioesters, as well as proteins with N-terminal Cys residues, to facilitate expressed protein ligation. As such, a more detailed understanding of the function of inteins can have significant impact for biotechnology applications. Here, we provide biochemical methods to study splicing activity and NMR methods to study intein structure and the catalytic mechanism.
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Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage.
Biochemistry, 2017Co-Authors: Christopher J. Minteer, Julie N. Reitter, Chunyu Wang, Robert J. Linhardt, Nicolle M. Siegart, Kathryn M. Colelli, Xinyue Liu, Alvin 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 an...
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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, Chunyu Wang, Robert J. Linhardt, Nicolle M. Siegart, Kathryn M. Colelli, 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
Biochemistry, 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, Chunyu Wang, 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
Tom W Muir - One of the best experts on this subject based on the ideXlab platform.
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Improved protein splicing using embedded split inteins
Protein science : a publication of the Protein Society, 2018Co-Authors: Josef A. Gramespacher, Adam J. Stevens, Robert E. Thompson, Tom W MuirAbstract:Naturally split inteins mediate a traceless protein ligation process known as protein trans-splicing (PTS). Although frequently used in protein engineering applications, the efficiency of PTS can be reduced by the tendency of some split intein fusion constructs to aggregate; a consequence of the fragmented nature of the split intein itself or the polypeptide to which it is fused (the Extein). Here, we report a strategy to help address this liability. This involves embedding the split intein within a protein sequence designed to stabilize either the intein fragment itself or the appended Extein. We expect this approach to increase the scope of PTS-based protein engineering efforts.
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A promiscuous split intein with expanded protein engineering applications
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Adam J. Stevens, David Cowburn, Neel H. Shah, Giridhar Sekar, Anahita Z. Mostafavi, Tom W MuirAbstract:The protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an “Extein dependence,” whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal Extein dependence. The resulting “promiscuous” inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins.
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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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Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-Splicing
2015Co-Authors: Neel H. Shah, David Cowburn, Ertan Eryilmaz, Tom W MuirAbstract: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 that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity
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Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-Splicing
Journal of the American Chemical Society, 2013Co-Authors: Neel H. Shah, David Cowburn, Ertan Eryilmaz, Tom W MuirAbstract: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 the...
Henning D. Mootz - One of the best experts on this subject based on the ideXlab platform.
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A functional interplay between intein and Extein sequences in protein splicing compensates for the essential block B histidine.
Chemical science, 2018Co-Authors: Kristina Friedel, Julian C. J. Matern, Popp, Emerich-mihai Gazdag, Ilka V. Thiel, Gerrit Volkmann, Wulf Blankenfeldt, Henning D. MootzAbstract:Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (−1) Extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(−1) residue, including D-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N–S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (−1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(−1)Phe and Gly(−1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the Extein–intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an Extein residue in protein splicing. It sheds new light on the Extein–intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins.
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Altered Coordination of Individual Catalytic Steps in Different and Evolved Inteins Reveals Kinetic Plasticity of the Protein Splicing Pathway
Journal of the American Chemical Society, 2018Co-Authors: Julian C. J. Matern, Kristina Friedel, Jens Binschik, Kira-sophie Becher, Zahide Yilmaz, Henning D. MootzAbstract:Protein splicing performed by inteins provides powerful opportunities to manipulate protein structure and function, however, detailed mechanistic knowledge of the multistep pathway to help engineering optimized inteins remains scarce. A typical intein has to coordinate three steps to maximize the product yield of ligated Exteins. We have revealed a new type of coordination in the Ssp DnaB intein, in which the initial N–S acyl shift appears rate-limiting and acts as an up-regulation switch to dramatically accelerate the last step of succinimide formation, which is thus coupled to the first step. The structure–activity relationship at the N-terminal scissile bond was studied with atomic precision using a semisynthetic split intein. We show that the removal of the Extein acyl group from the α-amino moiety of the intein’s first residue is strictly required and sufficient for the up-regulation switch. Even an acetyl group as the smallest possible Extein moiety completely blocked the switch. Furthermore, we inv...
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Altered Coordination of Individual Catalytic Steps in Different and Evolved Inteins Reveals Kinetic Plasticity of the Protein Splicing Pathway
2018Co-Authors: Julian C. J. Matern, Kristina Friedel, Jens Binschik, Kira-sophie Becher, Zahide Yilmaz, Henning D. MootzAbstract:Protein splicing performed by inteins provides powerful opportunities to manipulate protein structure and function, however, detailed mechanistic knowledge of the multistep pathway to help engineering optimized inteins remains scarce. A typical intein has to coordinate three steps to maximize the product yield of ligated Exteins. We have revealed a new type of coordination in the Ssp DnaB intein, in which the initial N–S acyl shift appears rate-limiting and acts as an up-regulation switch to dramatically accelerate the last step of succinimide formation, which is thus coupled to the first step. The structure–activity relationship at the N-terminal scissile bond was studied with atomic precision using a semisynthetic split intein. We show that the removal of the Extein acyl group from the α-amino moiety of the intein’s first residue is strictly required and sufficient for the up-regulation switch. Even an acetyl group as the smallest possible Extein moiety completely blocked the switch. Furthermore, we investigated the M86 intein, a mutant with faster splicing kinetics previously obtained by laboratory evolution of the Ssp DnaB intein, and the individual impact of its eight mutations. The succinimide formation was decoupled from the first step in the M86 intein, but the acquired H143R mutation acts as a brake to prevent premature C-terminal cleavage and thereby maximizes splicing yields. Together, these results revealed a high degree of plasticity in the kinetic coordination of the splicing pathway. Furthermore, our study led to the rational design of improved M86 mutants with the highest yielding trans-splicing and fastest trans-cleavage activities
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An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site.
The Journal of biological chemistry, 2015Co-Authors: Anne-lena Bachmann, Henning D. MootzAbstract:Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the Extein-N and Extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine, or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal uphill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N terminus. The Int(N) fragment could be shortened from 37 to 28 amino acids and exchanged with the 25-amino acid Int(N) fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the Int(C) fragment of the GOS-TerL intein.
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Structure-activity studies on the upstream splice junction of a semisynthetic intein.
Bioorganic & medicinal chemistry, 2013Co-Authors: Alexandra Wasmuth, Christina Ludwig, Henning D. MootzAbstract:Abstract Protein trans -splicing by split inteins holds great potential for the chemical modification and semisynthesis of proteins. However, the structural requirements of the Extein sequences immediately flanking the intein are only poorly understood. This knowledge is of particular importance for protein labeling, when synthetic moieties are to be attached to the protein of interest as seamlessly as possible. Using the semisynthetic Ssp DnaB intein both in form of its wild-type sequence and its evolved M86 mutant, we systematically varied the sequence upstream of the short synthetic Int N fragment using both proteinogenic amino acids and unnatural building blocks. We could show for the wild-type variant that the native N-Extein sequence could be reduced to the glycine residue at the (−1) position directly flanking the intein without significant loss of activity. The glycine at this position is strongly preferred over building blocks containing a phenyl group or extended alkyl chain adjacent to the scissile amide bond of the N-terminal splice junction. Despite their negative effects on the splicing yields, these unnatural substrates were well processed in the N–S acyl shift to form the respective thioesters and did not result in an increased decoupling of the asparagine cyclization step at the C-terminal splicing junction. Therefore, the transesterification step appeared to be the bottleneck of the protein splicing pathway. The fluorophore 7-hydroxycoumarinyl-4-acetic acid as a minimal N-Extein was efficiently ligated to the model protein, in particular with the M86 mutant, probably because of its higher resemblance to glycine with an aliphatic c-α carbon atom at the (−1) position. This finding indicates a way for the virtually traceless labeling of proteins without inserting extra flanking residues. Due to its overall higher activity, the M86 mutant appears most promising for many protein labeling and chemical modification schemes using the split intein approach.
Marlene Belfort - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing.
Biochemistry, 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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Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership.
mBio, 2018Co-Authors: Christopher W. Lennon, Matthew J. Stanger, Nilesh K. Banavali, Marlene BelfortAbstract:ABSTRACT 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. IMPORTANCE Inteins are mobile genetic elements that interrupt coding sequences (Exteins) and are removed by protein splicing. They are abundant elements in microbes, and recent work has demonstrated that protein splicing can be controlled by environmental cues, including the substrate of the intein-containing protein. Here, we describe an intein-Extein collaboration that controls temperature-induced splicing of RadA from two archaea and how variation in this intein-Extein partnership results in fine-tuning of splicing to closely match the environment. Specifically, we found that a small sequence difference between the two inteins, a flexible loop that likely once housed a homing endonuclease used for intein mobility, acts as a switch to control intein-Extein interactions that block splicing. Our results argue strongly that some inteins have evolved away from a purely parasitic lifestyle to control the activity of host proteins, representing a new form of posttranslational regulation that is potentially widespread in the microbial world.
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Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership
American Society for Microbiology, 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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Additional file 3: of The dynamic intein landscape of eukaryotes
2018Co-Authors: Cathleen Green, Olga Novikova, Marlene BelfortAbstract:Inteins in vDNA. This is a spreadsheet of eukaryotic inteins in vDNA with accession numbers and Extein/intein sequences mined in this study. (XLSX 80Â kb
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post translational environmental switch of rada activity by Extein intein interactions in protein splicing
Nucleic Acids Research, 2015Co-Authors: Natalya I. Topilina, Nilesh K. Banavali, Matthew J. Stanger, O S Novikova, Marlene BelfortAbstract:Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the Exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native Exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote Extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native Exteins and the intein, in three-dimensional space. The Exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its Exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.
