2018
DOI: 10.1021/jacs.8b04794
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Altered Coordination of Individual Catalytic Steps in Different and Evolved Inteins Reveals Kinetic Plasticity of the Protein Splicing Pathway

Abstract: 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 acce… Show more

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Cited by 11 publications
(7 citation statements)
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“…Our observations would be consistent with all (–1) residues larger than the native Gly(–1) rendering the Ex N –Int N complexes unstable (complex I) under these conditions and that the Coomassie-stained SDS gels only showed the cleaved complexes with the Int N peptide (complex II). These differences in complex stability would be in agreement both with the higher fragment affinity of the M86 mutant in the case of the split inteins24 and with the higher thermostability of the cis -M86 intein compared to that of the cis -WT intein 31. However, since the SDS-PAGE based assays shown in Fig.…”
Section: Resultssupporting
confidence: 59%
“…Our observations would be consistent with all (–1) residues larger than the native Gly(–1) rendering the Ex N –Int N complexes unstable (complex I) under these conditions and that the Coomassie-stained SDS gels only showed the cleaved complexes with the Int N peptide (complex II). These differences in complex stability would be in agreement both with the higher fragment affinity of the M86 mutant in the case of the split inteins24 and with the higher thermostability of the cis -M86 intein compared to that of the cis -WT intein 31. However, since the SDS-PAGE based assays shown in Fig.…”
Section: Resultssupporting
confidence: 59%
“…Given that thioesters should be cleaved efficiently by β‐ME we believe this band represents the second (oxoester) branched intermediate, which is in chemical equilibrium with the first thioester intermediate. The transient build‐up of the branched intermediate has previously been observed for other inteins and is indicative of asparagine cyclization as the last step of protein splicing being rate determining for the entire splicing pathway [6a,c,13] …”
Section: Resultsmentioning
confidence: 65%
“…The M86 intein is more resistant to thermal unfolding than its parent and promotes step 1 (amide-to-ester or -thioester rearrangement) in protein splicing while slowing step 3 of splicing (Asn cyclization). 40 Although the mutations to the EXH motif of the P. abyssi PolII intein result in the loss of activity rather than improved structural stability and activity, our results suggest that the EXH itself promotes structural stability of the intein fold, despite its distance from the active site. It would be interesting to determine if inserting an EXH motif into an intein that lacks it could increase intein resistance to denaturation at high temperatures or salt concentrations; however, the lack of overall sequence conservation in the EXH may make this challenging.…”
Section: ■ Discussionmentioning
confidence: 64%
“…The authors report that the selected mutations appear to be additive and that at least two mutations are on the opposite face of the intein, and two others in the loop where the homing endonuclease would be are far from the active site. The M86 intein is more resistant to thermal unfolding than its parent and promotes step 1 (amide-to-ester or -thioester rearrangement) in protein splicing while slowing step 3 of splicing (Asn cyclization) . Although the mutations to the EXH motif of the P. abyssi PolII intein result in the loss of activity rather than improved structural stability and activity, our results suggest that the EXH itself promotes structural stability of the intein fold, despite its distance from the active site.…”
Section: Discussionmentioning
confidence: 73%