V67L Mutation Fills an Internal Cavity To Stabilize RecA <i>Mtu</i> Intein

Zwarycz, Allison S.; Fossat, Martin J.; Akanyeti, Otar; Lin, Zhongqian; Rosenman, David J.; Garcı́a, Angel E.; Royer, Catherine A.; Mills, Kenneth V.; Wang, Chunyu

doi:10.1021/acs.biochem.6b01264

Cited by 10 publications

(9 citation statements)

References 44 publications

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“…Previous studies on intein engineering have mostly focused on the conserved residues or neighboring exteins, this study suggests the feasibility of engineering controllable inteins by mutating the residues surrounding the conserved ones. As a further perspective, following the recent development of photocaged cysteine, tyrosine, and serine amino acid residues which have been introduced in living systems, light activated C‐terminal cleavage inteins may be obtained by incorporating these and other photocaged residues.…”

Section: Discussionmentioning

confidence: 86%

Engineered pH‐inducible intein Mtu ΔI‐CM variants with markedly reduced premature cleavage activity

et al. 2019

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Inteins have been widely exploited for the purification of tagless proteins. Among them, pH‐inducible C‐terminal‐cleavage inteins enable the preparation of proteins and peptides with an authentic N‐terminus. However, a severe premature cleavage around neutral pH has limited the application of these inteins, especially when used in recombinant hosts such as Escherichia coli. By targeting the microenvironment of the two key histidine residues H429 and H439, we engineered Mtu ΔI‐CM intein to markedly reduce its premature cleavage. Kinetic analyses suggested that although the variants retained the pH dependence, they indeed cleaved slower, especially at pH 7.6. These variants resulted in higher yields for two model polypeptides than the original Mtu ΔI‐CM intein, when used in conjunction with a cleavable self‐assembling tag. This work suggests that more controllable pH‐inducible inteins can be obtained by manipulating the residues in the self‐cleavage sites and provide better performance for tag‐based protein preparation strategies.

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Section: Discussionmentioning

confidence: 86%

Engineered pH‐inducible intein Mtu ΔI‐CM variants with markedly reduced premature cleavage activity

et al. 2019

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“…57 Biophysical characterization of the evolved mini-inteins suggests that the selected mutations are important to restore the structural stability of the intein overall while concomitantly restoring flexibility near the site of the deletion. [57][58][59][60][61][62] The Synechocystis sp. PCC6803 DnaB mini-intein was developed by deletion of its native HEN, with directed evolution experiments improving activity and flexibility of the intein insertion site.…”

Section: Resultsmentioning

confidence: 99%

“…The Mycobacterium tuberculosis RecA intein has a native HEN domain but has been engineered to splice in trans and to function as a mini-intein. , However, protein splicing of the engineered mini-intein was inefficient and required directed evolution experiments to optimize activity . Biophysical characterization of the evolved mini-inteins suggests that the selected mutations are important to restore the structural stability of the intein overall while concomitantly restoring flexibility near the site of the deletion. − The Synechocystis sp. PCC6803 DnaB mini-intein was developed by the deletion of its native HEN, with directed evolution experiments improving the activity and flexibility of the intein insertion site. , Deletion of the HEN domain is deleterious in a variant of the Methanococcus jannaschii TFIIB intein, in which the part of the loop that replaces the HEN domain inserts as a β-strand into the HINT domain; the authors suggest that the native HEN domain could facilitate the proper folding of the pseudosubdomains of the HINT domain .…”

Section: Resultsmentioning

confidence: 99%

Protein Splicing Activity of the Haloferax volcanii PolB-c Intein Is Sensitive to Homing Endonuclease Domain Mutations

et al. 2020

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Inteins are selfish genetic elements residing in open reading frames that can splice posttranslationally, 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 Å separation in a PolB intein homology model. The combination of in vivo and in vitro evidence strongly supports a model of co-dependence 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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“…The authors observed that the V67L mutation did not affect the overall conformation but claim that it improved side chain packing . The suggestion that the bulkier side chain filled an internal cavity was further supported by high-pressure NMR measurements of the cavity volume change due to the V67L mutation, 13 ± 3 mL mol –1 , roughly corresponding to the size of a methylene group . The mutation demonstrated global NMR chemical shift perturbations, including active site residues, and slowed hydrogen–deuterium exchange, suggesting that global conformational dynamics were reduced .…”

Section: Discussionmentioning

confidence: 97%

“…27 The suggestion that the bulkier side chain filled an internal cavity was further supported by high-pressure NMR measurements of the cavity volume change due to the V67L mutation, 13 ± 3 mL mol −1 , roughly corresponding to the size of a methylene group. 35 The mutation demonstrated global NMR chemical shift perturbations, including active site residues, and slowed hydrogen−deuterium exchange, suggesting that global conformational dynamics were reduced. 36 This is in agreement with a recent computational study of the V67L intein, 37 where V67L reduced conformational fluctuation to various extents.…”

Section: ■ Discussionmentioning

confidence: 99%

Allosteric Influence of Extremophile Hairpin Motif Mutations on the Protein Splicing Activity of a Hyperthermophilic Intein

et al. 2020

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Protein splicing is a post-translational process mediated by an intein, whereby the intein excises itself from a precursor protein with concomitant ligation of the two flanking polypeptides. The intein that interrupts the DNA polymerase II in the extreme hyperthermophile Pyrococcus abyssi has a β-hairpin that extends the central βsheet of the intein. This β-hairpin is mostly found in inteins from archaea, as well as halophilic eubacteria, and is thus called the extremophile hairpin (EXH) motif. The EXH is stabilized by multiple favorable interactions, including electrostatic interactions involving Glu29, Glu31, and Arg40. Mutations of these residues diminish the extent of Nterminal cleavage and the extent of protein splicing, likely by interfering with the coordination of the steps of splicing. These same mutations decrease the global stability of the intein fold as measured by susceptibility to thermolysin cleavage. 15 N− 1 H heteronuclear single-quantum coherence demonstrated that these mutations altered the chemical environment of active site residues such as His93 (B-block histidine) and Ser166 (F-block residue 4). This work again underscores the connected and coordinated nature of intein conformation and dynamics, where remote mutations can disturb a finely tuned interaction network to inhibit or enhance protein splicing.

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V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein

Cited by 10 publications

References 44 publications

Engineered pH‐inducible intein Mtu ΔI‐CM variants with markedly reduced premature cleavage activity

Engineered pH‐inducible intein Mtu ΔI‐CM variants with markedly reduced premature cleavage activity

Protein Splicing Activity of the Haloferax volcanii PolB-c Intein Is Sensitive to Homing Endonuclease Domain Mutations

Allosteric Influence of Extremophile Hairpin Motif Mutations on the Protein Splicing Activity of a Hyperthermophilic Intein

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