Altered Coordination of Individual Catalytic Steps in Different and

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Altered coordination of individual catalytic steps in different and evolved inteins reveals kinetic plasticity of the protein splicing pathway Julian C. J. Matern, Kristina Friedel, Jens Binschik, Kira-Sophie Becher, Zahide Yilmaz, and Henning D. Mootz J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04794 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 2018

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Journal of the American Chemical Society

Altered coordination of individual catalytic steps in different and evolved inteins reveals kinetic plasticity of the protein splicing pathway Julian C. J. Matern, Kristina Friedel, Jens Binschik, Kira-Sophie Becher, Zahide Yilmaz, Henning D. Mootz* Institute of Biochemistry, Department of Chemistry and Pharmacy, University of Muenster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany. ABSTRACT: Protein splicing performed by inteins provides powerful opportunities to manipulate protein structure and function, however, detailed mechanistic knowledge of the multi-step 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 semi-synthetic 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.

Inteins are internal protein domains that are translated as part of a precursor protein between flanking N- and Cterminal extein sequences.1-2 They undergo a spontaneous self-processing reaction termed protein splicing, in which they excise themselves out of the precursor with concomitant ligation of the extein sequences to reconstitute the host protein.3-4 As tools to alter the peptide backbone of proteins, inteins have found many applications in the fields of biotechnology, protein biochemistry, chemical biology, and synthetic biology.5-6 For these purposes, the identification and engineering of inteins with superior activity is a pressing goal, for example in terms of yield, rate and sequence tolerance and among other features.7-17 However, our understanding of the coordination in the multi-step protein splicing pathway is very scarce for most inteins and therefore limits our ability to design tailor-made inteins for peptide-bond making and breaking applications.

second step, the ExN is transferred by a transesterification reaction onto the side chain of a Cys (or Ser/Thr) residue at the +1 position, the first amino acid of the C-extein (ExC), to give a branched intermediate. In the third step, the side chain amide of the Asn residue at the last position of the intein attacks the C-terminal scissile bond to break it by forming itself a succinimide. This step is considered irreversible, in contrast to the first two reversible steps. Finally, in an uncatalyzed S-N (or O-N) acyl shift involving the liberated α-amine group of the +1 residue, the thioester (or oxoester) between the two exteins rearranges to the stable peptide bond and thereby completes formation of the mature host protein. Inteins are also known in split form, either naturally occurring or artificially engineered.19-23 Split inteins catalyze protein transsplicing, by which the extein sequences are joined from two separate polypeptides through the same individual reactions and mechanistic principles, however, involving a preceding step of intein fragment association.

Inteins can be regarded as single turn-over catalysts for the first three reactions of the four-step canonical protein splicing pathway (Figure 1).3, 18 In the first step, the Nterminal scissile peptide bond is rearranged in an N-S (or N-O) acyl shift of the N-extein sequence (ExN) onto the nucleophilic side chain of a Cys (or Ser) at position 1 of the intein to give a linear (thio)ester intermediate. In the

In order to achieve high protein splicing yields, inteins have to optimize the coherence of the three catalyzed steps by adapting kinetic rates or by functional coupling. The linear and branched (thio)ester intermediates can be prone to cleavage by hydrolysis or attack of other nucleophiles leading to cleavage of the N-terminal scissile bond, referred to as N-terminal cleavage. A premature succin-

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imide formation leads to C-terminal cleavage. An early example of functionally coupled steps in the pathway was discovered by mutation of the key Cys1 at the N-terminal splice junction in the homologous Ssp and Npu DnaE split inteins, which also blocked C-terminal succinimide formation.8, 24 When leaving the Cys1 unmutated, a chemically induced nucleophilic cleavage of the linear thioester intermediate triggered C-terminal cleavage by succinimide formation in the Ssp DnaE intein. The N-S acyl shift was at least ten times faster than overall splicing, indicating that the transthioesterification and/or succinimide formation reaction represents the rate-limiting step(s) of the Ssp DnaE intein (Figure 1A).24-25

Most other inteins investigated, including the Ssp DnaB intein from Synechocystis spp. PCC6803,29 likewise do not require cleavage at the N-terminal scissile bond for succinimide formation, as they are able to perform Cterminal cleavage in the presence of a C1A mutation.30-31 Nevertheless, previous studies by us and others showed that succinimide formation of the Ssp DnaB intein is coupled to the N-terminal splice junction, because it is greatly accelerated when the ExN is missing, compared to a C1A mutant including the ExN moiety.32-33 These observations indicated that the protein splicing pathway of the Ssp DnaB intein might employ a third type of kinetic regulation and functional coupling and called for the detailed investigation in this study.

To investigate the pathway starting from a defined intermediate is more difficult and was accomplished by only one study so far. Muir and co-workers revealed a different type of kinetic regulation and functional coupling in the Mxe GyrA intein (Figure 1B).26 A semisynthetic branched intermediate was prepared by expressed protein ligation and shown to be competent in protein splicing after refolding, employing a C1S substitution to minimize reversion into the linear intermediate. The resolution rate of the branched intermediate by succinimide formation was found to be very similar to the overall splicing reaction and about 50-fold slower than the initial N-S shift. These results suggested that succinimide formation from the branched intermediate is the rate-limiting step for the Mxe GyrA intein, in agreement with previous observations.27 Importantly, the rates of succinimide formation from both a mutated linear precursor (with a C1A mutation to block the upstream scissile bond) and a linear variant lacking the ExN sequence were about 10-fold lower, indicating a functional coupling that up-regulates succinimide formation upon formation of the branched intermediate. In contrast to the Ssp DnaE intein,24, 28 the rate of succinimide formation was insensitive to changes at the N-terminal splice junction.26 A

Ssp & Npu DnaE intein (previous work) fast N

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We also asked the question how the evolution of an intein will affect the three individual reactions when the selective pressure was to maximize the yield or rate of the overall pathway? Mutations known to increase the rate of splicing in the Ssp and Npu DnaE inteins were shown to differentially affect their individual reactions, yet no functional couplings related to intermediates of the splicing pathway were investigated in these cases.9, 16, 34 Single and double mutants of the Mtu RecA intein were created by directed evolution and shown to act synergistically to increase both splicing and C-terminal cleavage rates. However, also in this case no potential functional coupling and possible change of the kinetics were investigated.10 Here we focussed on the Ssp DnaB intein and its M86 mutant, which was previously isolated from a sequential directed evolution experiment by selection for mutants with higher splicing activity in different sequence contexts.11 The M86 intein contained eight mutations. In artificially split versions using a short intein-N (IN) fragment of only 11 aa,35 the split M86 intein is higher yielding and up to ~60-fold faster than the parent split Ssp DnaB intein.11 These inteins have been explored as powerful tools for protein engineering, for example using a synthetic ExN-IN fragment for N-terminal chemical pro-

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tein labeling.11, 36-37 Figure 1. Kinetic models of the protein splicing pathway for the A) Ssp & Npu DnaE, B) Mxe GyrA and C) Ssp DnaB inteins. Reactions 1-3 are intein-catalyzed, whereas reaction 4 occurs spontaneously. Note that the Ssp & Npu DnaE inteins are split inteins but are depicted here as cis-inteins for reasons of clarity. Arrows indicate the up-regulation switch with the acceleration factor for the rate of succinimide formation after formation of the branched intermediate in case of the 26 Mxe GyrA intein (B), and the linear thioester intermediate in case of the Ssp DnaB intein (C). For the latter the factor of 50-160 represents the range of values determined for peptides pep2-pep5 and pep7. N = N-extein; I = intein; C = C-extein.

Here we report a detailed analysis on the structural changes at the N-terminal splice junction of the Ssp DnaB intein that trigger accelerated succinimide formation. We show that this intein exhibits a third type of coordination of the splicing pathway with the first step of N-S acyl transfer being rate-determining. Our comparative study with the M86 intein and the individual impact of its 8 mutations revealed an even further-reaching kinetic plasticity of the splicing pathway. The coordination of this intein has changed as a result of the laboratory evolution and is more similar to the Mxe GyrA intein with succinimide formation representing the rate-determining step. RESULTS The N-S acyl shift as an up-regulation switch of succinimide formation in the Ssp DnaB intein We previously observed that the Ssp DnaB mini-intein38 from Synechocystis sp. PCC6803 underwent rapid succinimide formation when the ExN was removed.32 We had exploited this phenomenon for a light-triggered transcleavage device to turn on protein activity upon unmasking its native N terminus. Here we asked whether this observation indicated a regulatory switch by coupling the initial N-S acyl shift with the succinimide formation. To understand the underlying mechanism in detail, we synthesized a set of ExN-IN peptides with structural variations around the N-terminal splice junction (Figure 2 and Table 1). The IN fragment encompassed amino acids 1-11 of the intein, while the IC counterpart (amino acids 12-154 of the wild-type (WT) Ssp DnaB mini-intein)36 was prepared recombinantly as a fusion protein with thioredoxin (Trx) as the C-terminal extein (WT-IC-Trx = construct 1). Figure 2 shows that succinimide formation in this semi-synthetic split version of the Ssp DnaB intein can be triggered in two different ways. First, pep1 with a C1A substitution to block the N-S acyl shift can promote C-terminal cleavage in the reconstituted split intein; however, the reaction rate is slow. Second, pep2, which lacks the ExN sequence, triggered a ~52-fold faster rate of C-terminal cleavage, consistent with previous observations.32 To untangle potential changes in the association step from the observed rate differences, we determined kinetic constants of complex formation. Changes in fluorescence anisotropy were measured upon incubation of the fluorescently labeled IN

peptides with protein WT-IC-Trx_AAA (construct 2; Figure S1). The H73A, N154A and S+1A mutations in this protein block all steps of the splicing pathway that follow intein fragment association.37 As shown in Figure 2D and Table 1, pep2 displayed a ~1.5-fold higher kon rate and a 2.8-fold lower Kd than pep1. These slight changes did not seem to sufficiently explain the ~52-fold difference in the rate of succinimide formation, in particular because concentrations of IN and IC partners in these reactions were 20 µM and therefore well above the Kd values of around 1 µM. The kinetic parameters of pep3, which combined the lacking ExN and the C1A substitution, were even more telling. Comparable kon and Kd values, 1.2-fold higher and 3.5-fold lower compared to pep1, respectively (Figure S1), were observed along with a 160-fold higher rate in succinimide formation (Figure 2D; see Figure S2 for SDS gels of all remaining peptides). We hypothesized that the peptides lacking the ExN mimicked the structure of the linear intermediate formed after the N-S acyl shift, with respect to the free α-amine group at Cys1. The higher rate of succinimide formation observed for pep2 would be coupled to the formation of the linear thioester intermediate in the first step of the splicing pathway and represent an up-regulation of the third step. To mimick the linear thioester intermediate more closely and to unravel structural features required for up-regulation in more detail, peptides pep4 to pep7 were designed (Figure 2B and Table 1). A 120-fold higher rate of succinimide formation was also found for pep4, a stable isoster of the linear thioester intermediate, further lending support to our hypothesis. Reducing the ExN sequence to an acetyl group in pep5, the smallest possible acyl ExN moiety, resulted in the similarly fast succinimide formation (148-fold faster than for pep1), indicating that the size of the acyl moiety on the side chain is irrelevant. In sharp contrast, when the acetyl group as ExN sequence was localized on the α-amino group in pep6, resembling the structure prior to the N-S acyl shift, only slow kinetics similar to pep1 were observed (no succinimide formation detectable in the first 4 h of the reaction; Figure 2D and Table 1). Furthermore, efficient up-regulation of succinimide formation was also observed with pep7 lacking the α-amine group, indicating that this moiety, once liberated by the N-S acyl shift, does not play an important role in the mechanism (Figure 2D and Table 1). Importantly, all these peptides showed very similar parameters for intein fragment association, indicating that the rate differences of succinimide formation resulted from consequences of the structural changes on the actual protein splicing pathway, at least mostly, and not on intein fragment association. Together, these findings showed a ~52-160 fold upregulation of succinimide formation in a split Ssp DnaB intein for an IN lacking its ExN linked to the α-nitrogen compared to the C1A mutant. Therefore, we see our hypothesis confirmed and propose that the linear thioester intermediate formed in the N-S acyl shift triggers the upregulation. This is a different mechanism than described for the Mxe GyrA intein, which showed up-regulation of



succinimide formation only in presence of the branched intermediate.26

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$ = values taken from Ref , given is ksplicing instead of kcleavage; n.d. = not detectable.

The N-S acyl shift is faster in the evolved M86 intein than in the parent Ssp DnaB intein

Figure 2. Rate of succinimide formation as a function of the structure around the N-terminal scissile bond. A) Scheme of the reactions. B) Structures around the scissile amide bond. Fl = 5,6-carboxyfluorescein. C) Coomassie-stained SDS-PAGE N C gels. I peptides and WT-I -Trx were incubated at 20 µM each. See Figure S2 for gels of reactions with pep3-pep7. Asterisks denote protein impurities. D) Rate of succinimide formation that were calculated from densitometric analyses of initial 4h of the reactions shown in C). n.d. = not detected.

Comparison of the rates of succinimide formation after the up-regulation switch with the rate of protein transsplicing37 showed that step 3 of the pathway was ~16-50 fold faster than the overall splicing pathway. The latter reaction was previously measured37 with the splicingcompetent IN peptide pep8 (compare entries for pep2pep5 and pep7 with pep8 in Table 1). In contrast, aberrant succinimide formation before the up-regulation switch, as measured with pep1, was slower than the overall process. From these observations we concluded that the N-S acyl shift had to be the rate-determining step of the Ssp DnaB intein. The transesterification reaction (step 2) has to be fast enough to ensure the covalent connection between the exteins before succinimide formation occurs and therefore cannot account for the ratedetermining step. Given that the evolved M86 mutant intein displays higher overall rates in protein splicing than the parent Ssp DnaB intein,11 these considerations predicted that it exhibited an accelerated N-S acyl shift. To test this assumption and to assay the N-S acyl shift, we used both cis mini-inteins in constructs MBP-WTintein(S+1I)-Trx (construct 3) and MBP-M86-intein(S+1I)Trx (construct 4), respectively. The S+1I mutation blocks the trans-esterification step and allowed the purification of the protein precursors in stable form.29 Cleavage of the linear thioester intermediate was then assayed in presence of 100 mM Mesna as a nucleophile (Figure 3). The rate of thiolysis should correlate with the rate of the N-S acyl shift. Cleavage rates were determined by densitometric analysis of precursor consumption and fitting to an exponential decay function (Figure 3C). They were found to be 5.2-fold higher for the M86 intein (kM86 = 5.0 ± 0.1 x 10-4 s-1; kwt = 0.96 ± 0.07 x 10-4 s-1), consistent with our model that the M86 precursor had to undergo a faster N-S acyl shift than the WT precursor.

Table 1. Kinetic parameters of IN peptides with WT-IC proteins

N

* Fl = 5,6-carboxyfluoresceine; I -part underlined; DAP = diaminopropionic acid; Ac = acetyl; # = the sequence of pep2a is identical with pep2 except for the lacking Fl moiety;


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Journal of the American Chemical Society formation of the linear thioester intermediate (compare M86 experiments with pep1 and pep2 in Figure 4). To test whether the faster N-S acyl shift and the slower succinimide formation in the M86 intein resulted in the expected higher population of the linear thioester, we intercepted this labile intermediate with high concentration of nucleophiles during protein splicing. Indeed, DTT at different concentrations had a more pronounced effect on the M86 intein than on the Ssp DnaB intein (Figure S3). Together, these findings revealed different relative rates and an altered coordination of the individual reactions in the splicing pathway of the evolved M86 intein. The M86 mutant displays increased thermal stability compared to the WT Ssp DnaB intein

Figure 3. Chemical cleavage of linear thioester intermediate. A) Scheme of the reactions using intein mutants with disabled C-terminal splice junction. Note that the intein-Trx product can undergo further cleavage into intein and Trx through succinimide formation. The asterisks denotes the S+1I mutation at the C-terminal splice junction. B) Analysis of reaction progress using Coomassie-stained SDS-PAGE gels. C) Time-courses of reactions determined from densitometric analyses of gels as shown in B).

To investigate the impact of the eight M86 mutations as a whole, we performed a thermal shift assay of the Ssp DnaB intein and the M86 mutant. Figure 5 shows that the M86 intein exhibited a significant increase of about 12 °C in thermostability in comparison to the WT Ssp DnaB intein (TmWT = 61.3 ± 0.7 °C; TmM86 = 73.4 ± 0.2 °C). These findings show that the acquired mutations of the M86 intein as a whole effect a higher thermal stability, which has co-evolved with the improved generality of the intein to splice with non-native exteins.

The evolved M86 intein displays an altered regulation of the individual steps in the protein splicing pathway The assays shown in Figure 3 revealed an additional difference between the Ssp DnaB and M86 cis-inteins. Following the chemical N-terminal cleavage of the precursor proteins into the respective ExN (MBP) and the remaining intein(S+1I)-Trx parts, the latter were cleaved into the intein and (S+1I)Trx pieces by succinimide formation. This subsequent reaction took place rapidly in case of the Ssp DnaB intein, in agreement with the up-regulation switch triggered by the lacking ExN, but was slower for the M86 intein (Figure 3B). To further investigate this notion, a trans-cleavage assay was performed with M86-IC-Trx (construct 5) of the split intein. Indeed, the slower Cterminal cleavage of the M86 intein compared to the Ssp DnaB intein was corroborated with peptide pep2a lacking the ExN sequence (Figure 4A). Interestingly, the M86 intein also showed robust trans-cleavage activity with pep1, in contrast to the parent Ssp DnaB intein, indicating succinimide formation was largely decoupled from the initial N-S acyl shift. The up-regulation switch was mostly lost as the rates displayed only a ~2.4-fold difference for the peptide mimicking the structures before and after

Figure 4. Decoupling of succinimide formation in the M86 intein. Reactions were performed as illustrated in Figure 2A, N however using an excess of the I peptides (30 µM) over the C I proteins (10 µM) to allow fitting of rates by pseudo firstorder kinetics. Note that pep2a is a derivative of pep2 that lacks the carboxyfluorescein moiety. A) Analysis of reactions using Coomassie-stained SDS-PAGE gels. Asterisks denote protein impurities. B) Reaction rates were calculated after fitting of time-courses determined from densitometric analyses of gels as shown in A).



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showed that the M86 mutations act additively and synergistically to improve the splicing activity, except for H143R, which decreased the rate of the succinimide formation and thereby balanced the kinetic coordination of the overall splicing pathway.

Figure 5. Thermostability of the Ssp DnaB intein (WT) and its M86 mutant. Both inteins were prepared as mini-inteins with five flanking extein residues on either side and inactivating mutations for all splicing and cleavage reactions at the key residues of the scissile bonds (C1A and N154A). A) Thermostability was determined by a thermal shift assay. B) Coomassie-stained SDS-gel of the proteins used in this assay.

Analysis of individual M86 mutations reveals key role of H143R mutation The evolved M86 intein had acquired eight mutations, of which seven are located at non-conserved positions and without direct contact to the active site, whereas one mutation (H143R) affected a conserved residue in the block F motif (Figure S4).11 The latter is believed to support asparagine cyclization by polarizing the side chain amide of the intein’s ultimate residue Asn154 through a charge relay system involving a neighboring water molecule.39 To understand the origin for the altered properties of the M86 intein, we studied the individual effects of the eight mutations. Eight derivatives of WT-IC-Trx (1) with one mutation each were prepared (constructs 6 to 13; Table S1). Note that all eight mutations of the M86 intein are located in the IC(12-154) fragment of the intein. In protein trans-splicing assays with the IN partner pep8, three of the mutations had a positive effect on the rate (I58T, S122P and P142L), two had a negligible effect (S18P and S107A) and three had a negative impact (D24G, S114P and H143R) (Figure 6A, Figure S5 and Table S2). Of the latter group, the H143R mutation in construct 13 led to a strongest rate decrease of about 4-fold. Strikingly, reversion of the H143R mutation in the M86 intein to give M86-IC(R143H)-Trx (construct 14) also reduced the yield of the protein trans-splicing reaction of the evolved mutant, despite an initially faster reaction. In the succinimide reaction triggered with pep2a as the IN peptide that lacks the ExN, seven out of eight of the single mutants (constructs 6 to 12) showed no significant differences compared to the unmutated WT-IC-Trx (1). Only the H143R substitution in construct 13 significantly slowed down the trans-cleavage reaction (~8-fold), resulting in a phenotype more similar to the M86 intein (Figure 6B, Figure S6 and Table S2). Reciprocally, the back mutation of this residue in the M86 intein, using M86IC(R143H)-Trx (14), resulted in the fastest succinimide reaction measured (~57-fold faster than for M86(5) and ~15-fold faster than for WT(1)). Together, these results

Surprisingly, a mutation of the key R143 in the M86 intein to alanine, giving mutant M86-IC(R143A)-Trx (construct 15), also led to a significantly faster succinimide formation than seen for the initial M86-IC-Trx (5), albeit not as fast as for M86-IC(R143H)-Trx (14) (Figure 6B). Likewise, the H143A mutation introduced into the parent Ssp DnaB intein (construct 16), had a lower impact on succinimide formation than H143R (Figure 6B), consistent with a previous report this mutation only partially impaired succinimide formation in the Ssp DnaB miniintein.39 These findings suggested that the key mutation H143R in the M86 intein not only slows down succinimide formation because of the loss of the catalytically involved histidine side chain, but that the arginine side chain actually acts as an active brake. In protein trans-splicing assayed in combination with pep8, the R143A mutant of M86 (construct 15) even exceeded the R143H mutant (14) in terms of splice product yields over C-terminal cleavage yields, despite a lower initial rate (Table S2 and Figure 6A). Obviously, the M86 intein with the R143A represents an even better balance between the rates of the individual reactions of the splicing pathway.

Figure 6. Kinetic rates of splicing and succinimide formation for split WT and M86 inteins. Grey columns denote


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reactions with WT intein or mutants thereof, black columns denote reactions with M86 intein or mutants thereof. A) Protein trans-splicing reactions were performed using pep8 C and the indicated I -Trx mutant constructs. B) C-terminal cleavage reactions by succinimide formation were carried out C using pep2a and the indicated I -Trx mutant constructs. C) and D) Analysis of protein trans-splicing reactions for the indicated M86 mutants. Shown are Coomassie-stained SDSPAGE gels. The asterisk denotes a protein contamination. See Figures S5 and S6 for gels of all reactions used for densitometric analysis. All values represented in the diagrams are given in Table S1.

The H143R mutation in the M86 intein favors accumulation of the branched intermediate On the basis of the altered reaction coordination in the evolved M86 intein we hypothesized that the deceleration of the succinimide formation might have decreased its rate close to becoming the rate-determining step. If so, an accumulation of the branched intermediate during splicing would be expected. This species has never been observed for the parent Ssp DnaB intein, a notion that is in agreement with our discovery of the up-regulation switch of succinimide formation for the parent intein. It also has not been observed for the trans-splicing reaction of the split M86 intein with fluorescent pep8.11 However, we noticed that the intein spliced somewhat slower with larger N-exteins, such as recombinant proteins. We prepared GFP-M86-IN (construct 17) and mixed it with M86IC-Trx (5). Indeed, an additional band was transiently detectable in the SDS-PAGE analysis during the course of the protein trans-splicing reaction (Figure 7A). The slower migration compared to the calculated size of the branched intermediate (Mcalc = 57.9 kDa) is likely due to the branched structure of the peptide backbone. Its identity could be further corroborated by Western blotting using an anti-GFP antibody. Consistent with our findings on the importance of Arg143, this intermediate band did not appear with the mutant M86-IC(R143H)-Trx (14), in which the key H143 residue was re-introduced to accelerate succinimide formation (Figure 7B). These findings further supported our model of altered kinetic regulation in the M86 intein and the impact of the H143R mutation.

Figure 7. Accumulation of branched intermediate (BI). Protein trans-splicing reactions were analyzed by SDS-PAGE

gels stained with Coomassie and immunoblotted using an N C anti-GFP antibody. A) Reaction of GFP-I (17) with M86-I N C Trx (5). B) Reaction of GFP-I (17) with M86(R143H)-I -Trx (14). A band at ~90 kDa was assigned to the BI (calculated: 57.9 kDa).

DISCUSSION Robust inteins with rapid and efficient splicing and cleavage activities are highly valuable tools for various applications in protein biotechnology and chemical biology, for example. A better understanding of the complex splicing pathway will be crucial for further identification and engineering of inteins with superior properties. New mutants arising on an evolutionary path to rapidly and efficiently splicing intein face the challenge to coordinate the individual steps to stay on-pathway by avoiding product loss through undesired side reactions. The challenge is most obvious for the succinimide formation step, because if occurring prematurely, the covalent connection between the exteins is irreversibly lost. It is therefore not surprising that for several inteins succinimide formation has been revealed as the overall ratedetermining step.40 This regulation serves as a safeguard to minimize premature C-terminal cleavage. For the Mxe GyrA intein it has been shown that this step is ratedetermining and functionally coupled to the formation of the branched intermediate (Figure 1B).26 In fact, on the basis of this consideration ultimately all inteins that manage to stay on-pathway must be characterized by a cleavage reaction of the C-terminal scissile bond that is regulated to only occur after branched intermediate formation. However, despite its importance, detailed biochemical knowledge on this regulatory mechanism in other inteins is very scarce, likely because in most studies the individual reactions were measured with intein mutants that are blocked in the other steps of the pathway. The use of such mutants may lead to false interpretations, because an up-regulation induced by an intermediate could be overlooked. In this study we have used structural analogs of the linear intermediate, either corresponding to IN peptides with the ExN entirely removed or shifted from the peptide backbone to the side chain in amide linkage as chemically stable isoster of the linear thioester. Our data suggests that for the Ssp DnaB intein succinimide formation is drastically up-regulated following the initial and ratedetermining N-S acyl shift (Figure 1C). Strikingly, after up-regulation the succinimide formation in the Ssp DnaB intein is significantly faster than the overall splicing rate (~16-fold; but must be rate-limiting step relative to the trans-esterification reaction to avoid loss of the ExN-ExC connection), indicating that the initial N-S acyl shift is the overall rate-determining step. In contrast, succinimide formation in the Mxe GyrA intein was found not to be upregulated by a cleavage of the N-terminal scissile bond.26 Even after up-regulation following formation of the branched intermediate, succinimide formation remained rate-determining for the overall rate of splicing of this



intein.26-27 The new type of coordination reported here for the Ssp DnaB intein is also distinct from that of the Ssp DnaE intein, since for the latter intein the N-S acyl shift was found not to be rate-limiting but 10-15 times faster than splicing (Figure 1A).24-25 Thus, we report here a third type of coordination of the protein splicing pathway in regular class 1 inteins. This new mechanism also makes sense under biochemical considerations, because it minimizes the life-time of the reactive (thio)ester intermediates and thereby the risk of their cleavage by cellular nucleophiles. Indeed, the thioester intermediate of the Ssp DnaB intein is known to be very prone to hydrolysis, in contrast to that of the Mxe GyrA intein, for example, and therefore calls for this precaution.29, 41 How is the kinetic coordination implemented in the protein splicing pathway of an intein? The coordination of selected individual reactions has previously been investigated with respect to sequential conformational changes and mechanistic couplings between single residues.42-43 Such changes on the molecular level will be required to control changes in rates. Further studies to elucidate them will help to understand the molecular mechanisms of the kinetic observations made here. Why is there such a high plasticity in the coordination of the protein splicing pathway? We propose that the presence of differentially coordinated inteins is unavoidable given the multistep nature of protein splicing and the common active site for all three catalyzed reactions. In their divergent evolution from a common ancestor, individual inteins have acquired various mutations in the presence of external cues (for example flanking sequence, temperature and required copy number of the mature host protein). It appears very unlikely that each acquired mutation would affect the rates of the three individual steps of protein splicing in a perfectly proportional fashion. Rather, a single mutation to improve the overall performance of the intein may change the coordination by changing the rate of one individual reaction relative to the other ones or imply differential changes on more than one reaction. Additionally, it may lead to the gain or loss of functional couplings between the reactions. Such changes will be tolerated and therefore carried further to the next generation as long as the overall outcome of splice product formation is sufficient and beneficial for host survival. By this mechanism different kinds of coordination of the individual steps may arise. Mutations must be avoided that would lead to unacceptable levels of a side-reaction based on the chemical cleavage of a labile intermediate, i.e., reactions of the ester intermediates with cellular nucleophiles. Additionally, the rate of the Cterminal cleavage step must be tightly linked to the rate of branched intermediate formation to act as a safeguard preventing premature and irreversible chain separation. This can be achieved in two basic ways: Either by an unconditional and sufficient overall decrease of the rate of succinimide formation relative to the preceding steps in the pathway (as described for the Mxe GyrA intein). Or by establishing a regulatory switch that keeps the rate of succinimide formation negligible until the pathway has

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reached an intermediate, and then gets up-regulated. The three types of coordination found in the Ssp DnaB, Mxe GyrA and Ssp DnaE inteins all employ an up-regulation switch of the second option (albeit in response to different intermediates). It should be noted, that in case of the Ssp DnaE intein the switch is only assumed to take place as inferred from cleavage assays of the linear thioester, but it was not shown directly from a defined intermediate or isoster thereof. The evolved M86 intein has mostly lost its up-regulation switch in response to the linear thioester intermediate, but we cannot rule out that it may have become sensitive to the branched intermediate. In fact, our kinetic analysis suggests that the directed protein evolution of the Ssp DnaB intein to the M86 intein, achieved through three consecutive rounds,11 can be seen as a representative blueprint for the alteration of the protein splicing pathway coordination by only a few mutations according to our proposal. We show that several of these mutations individually accelerated protein splicing but did not proportionally increase the rate of succinimide formation. The H143R mutation was acquired in the second round of directed evolution and slows down succinimide formation with a maximizing effect on product yields.11 As mentioned before, the coupled up-regulation switch of succinimide formation following the initial N-S acyl shift, observed in the parent Ssp DnaB intein, was mostly lost in the evolved M86 intein. Finally, our insights into the role of the H143R mutation allowed us to design the M86(R143A) mutant, which is the best M86 variant with regard to the combination of high yields and fast reaction kinetics under the conditions tested here. Furthermore, the designed M86(R143H) mutant was the fastest intein in this study in the C-terminal trans-cleavage reaction (14 and 57-fold faster than the Ssp DnaB and M86 inteins, respectively). These two findings nicely underline how improved mechanistic understanding of the protein splicing pathway can lead to engineered inteins with superior properties. EXPERIMENTAL SECTION Production and purification of proteins All constructs were expressed in E. coli BL21 DE3(DE3) gold grown in LB medium. Protein expression was induced at 28°C for 4 h by addition of either IPTG (0.4 mM) or arabinose (0.2 %), depending on the plasmid. Cell pellets were stored at -20°C until further use. Cells were resuspended in the respective ice-cold buffer and ruptured using an Emulsiflex C5 (Avestin) or a homogenizer in presence of 8 M urea (Potter-Elvehjem). The insoluble fraction was removed by centrifugation (20 min, 25000 rcf, 4°C). For proteins fused to a His6 tag, cells were resuspended in Ni-NTA buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) and proteins were purified by Ni-NTA affinity chromatography. Purification was either performed under native conditions at 4°C or under denaturing conditions at room temperature in the additional presence of urea (8 M) using gravity flow columns with a bed volume of typically 1.5 mL. Proteins were elut-


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ed with 250 mM imidazole. For proteins containing an MPB tag, cells were resuspended in amylose column buffer (ACB, 20 mM Tris-HCl, 200 mM NaCl, pH 7.4). Purification was performed using a gravity flow column with amylose material (New England Biolabs, typical bed volume 2 mL). Elution of the target protein was achieved with ACB containing maltose (2 mM). For proteins with an SBP or StrepII tag, cells were resuspended in buffer W (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8). The soluble fraction was applied twice onto a Strep-Tactin gravity-flow column (IBA, typical bed volume 1.5 mL). Elution of the target protein was achieved by addition of buffer W with desthiobiotin (2.5 mM). In all cases, fractions containing the purified protein were pooled and dialyzed three times against splice buffer (50 mM TrisHCl, 300 mM NaCl, 1 mM EDTA, pH 7) with DTT (2 mM) added in the first step. Glycerol (10 % v/v) was added prior to flash freezing of protein aliquots in liquid nitrogen and storage at -80°C. Protein concentrations were determined using the calculated extinction coefficients at 280 nm. Anion-exchange was performed on a Mono-Q PE 4.6/100 column at 4°C using an Äkta Purifier (GE Healthcare). The protein solution was dialyzed against MonoQ buffer A (20 mM Tris-HCL, pH 8.0) and applied to the column with a flow rate of 0.75 mL/min. The proteins were eluted with a gradient from 0 to 500 mM NaCl. Splice assays Inteins with individual mutations were assayed using the respective fusion protein WT-IC-Trx-H6 (1) or M86-IC-TrxH6 (5) (10 µM) and synthetic IN peptide pep8 (30 µM) in splice buffer with TCEP or DTT (2 mM) at 25°C. These conditions allowed a more stringent differentiation between the two inteins than those previously reported (15 µM and 75 µM, respectively).11 Aliquots were withdrawn at different time points, immediately mixed with 4x SDS sample buffer (containing β-mercapothanol) and stored at -20°C until further analysis. The samples were heated to 95°C for 10 min before analysis by SDS-PAGE. To evaluate the effect of DTT on the splice reaction, different DTT concentrations were used in a set of assays, using dilutions from a stock solution (1 M).

tography. While kept on ice, they were diluted to a final concentration of 10 µM using splice buffer. The assay was started by adding 2 mM TCEP and 100 mM MESNA and shifting the reaction mixture to a heating block at 25°C immediately. Aliquots were withdrawn at different time points, quenched by addition of 4x SDS sample buffer (without β-mercaptoethanol) and immediately stored at 20 °C until further analysis. Thermal shift assays As a fluorescent dye, a stock solution of 5000x SYPRO™ Orange was used. Protein solutions and SYPRO™ Orange where added to the wells of a 96-well PCR plate to give final concentrations of 5 mg/ml protein, 5x. The PCR plate was sealed and placed into the CFX96 Touch™ RealTime PCR Detection System (Bio-Rad). Samples where heated from 10 to 90°C in increments of 1°C while fluorescence was measured. The measurements for each protein were performed 10 times. The truncated fluorescence imaging data was normalized and fitted to a Boltzmann sigmoidal curve using GraphPad Prism 3: = +

− − 1 + exp

Densitometric analysis and calculation of rateconstants Evaluation of signal intensity of Coomassie-stained bands in SDS-PAGE analysis was done using the programs “Scion image” (Scion corporation) or “gel analyzer 2010 a” (gelanalyzer.com). For calculation of second-order rate constants in assays using the splicing partners at equimolar concentrations, the collected data was fitted to the following equation using “Origin” (OriginLab) or “QTIPlot” (www.qtiplot.com). 1 1 = + ∗

([P]T = substrate concentration at time t; [P]0 = initial substrate concentration; KRe = second-order reaction constant; t = reaction time) For experiments performed using an excess of one splice partner, the data was fitted to the following pseudo-first order equation using “QTI-Plot”.24 = ∗ 1 − !"#$%∗&' (

Succinimide-formation assays Equimolar concentrations of IN peptide and IC protein (20 µM each) were incubated at 25°C in splice buffer with 2 mM DTT. For pseudo-first order conditions, concentrations of 30 and 10 µM, respectively, were used. Aliquots were withdrawn and immediately quenched by addition of 4x SDS sample buffer with β-mercaptoethanol. The samples were stored at -20°C and heated to 95°C for ten minutes before further analysis. N-terminal thiolysis assays of cis-intein precursors Precursor proteins containing the S+1I mutation were purified by amylose affinity and anion-exchange chroma-

([P]T = substrate concentration at time t; [P]0 = initial substrate concentration; KRe = pseudo-first order reaction constant; t = reaction time) Detection of the branched intermediate GFP-IN-SBP (17; 10 µM) was pre-incubated for 5 min at 25°C in splice buffer with TCEP (2 mM). The reaction was started by adding the M86-IC-Trx-H6 (5) or its R143H variant (14) in equimolar amounts. Aliquots were drawn at different time points, immediately quenched by addition of 4x SDS sample buffer (without βmercaptoethanol) and stored at -20°C. Samples were analyzed by SDS-PAGE after heating to 95°C for 10 min.



For analysis by western blotting, an anti-GFP antibody (Covance, mouse, 1:10000) and a secondary anti-mouseantibody HRP-conjugate were used (Dako Diagnostica, rabbit, 1:5000).

ABBREVIATIONS N

C

Ex = N-extein; Ex = C-extein; IN = N-terminal intein C fragment, I = C-terminal intein fragment; WT = wild-type (Ssp DnaB intein).

REFERENCES

Determination of binding constants Fluorescence anisotropy was measured in 384-well microtiter plates using a SpectraMax M5 (Molecular Devices). Plate and machine were pre-incubated to 25°C for 30 min. The respective IN peptide solution (1 µL, 50 µM concentration) was mixed with protein 2 (49 µL) in the appropriate concentration range (2.5-30 µM). Subsequently, anisotropy was measured in a time-dependent fashion. To evaluate KD-values anisotropy-data was fitted to the equation: ) = )*+ +

Page 10 of 12

,!- + . + / ' − 0 !!- + . + / '1' − 4 ∗ . ∗ -(3 ∗ !)45 − )*+ ' !2 ∗ .'

(F = measured fluorescence anisotropy; Fmin = anisotropy of free peptide; Fmax = saturation anisotropy; KD = complex dissociation constant; C = protein concentration; L = peptide concentration) For calculation of Kon values the individual measurements were treated as first-order association reactions and fitted to equation (2). The resulting rate constants were plotted and fitted by linear regression to obtain the Kon from the slope. The Koff value can be obtained from the y-intercept. Peptide synthesis Solid-phase peptide synthesis was carried out as described previously.44

ASSOCIATED CONTENT Supporting Information. Contains supporting Tables and Figures as well as the experimental section. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources We are grateful for financial support for this work by the DFG (grant numbers MO1073/3-2 and MO1073/5-2 in the SPP1623 program).

The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Patrick Hansmann and Daniel Kümmel for help with the thermal shift assay.

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Altered Coordination of Individual Catalytic Steps in Different and

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