Conserved residues that modulate protein<i>trans</i>-splicing of<i>Npu</i>DnaE split intein

Wu, Qiulian; Gao, Zengqiang; Wei, Yong; Ma, Guolin; Zheng, Yuan; Dong, Yuhui; Liu, Yangzhong

doi:10.1042/bj20140287

Cited by 12 publications

(15 citation statements)

References 49 publications

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“…Lastly, the A136S mutation on Ssp C has previously been shown to accelerate protein splicing and was examined separately. 8 This A136S mutation increases the rate of branch resolution 2-fold, but has no impact on branch formation ( Figures S4C, S4D ).…”

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confidence: 97%

Design of a Split Intein with Exceptional Protein Splicing Activity

et al. 2016

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Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques.

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confidence: 97%

Design of a Split Intein with Exceptional Protein Splicing Activity

et al. 2016

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“…In addition, N Arg50 was shown to act positively on splicing . According to our structure N Arg50 could influence the orientation of N Cys1, C Asp17 and/or the extein's peptide backbone (Figures and S6).…”

Section: Figurementioning

confidence: 67%

Mechanistic Insights into Cyclic Peptide Generation by DnaE Split‐Inteins through Quantitative and Structural Investigation

et al. 2017

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Inteins carry out protein-splicing reactions, which are used in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in head-to-tail cyclisation of the target sequence, which can be used for genetic encoding and expression of libraries of cyclic peptides (CPs). The efficiency of the splicing reaction depends on the target sequence. Here we used mass spectrometry to assess in vivo cyclic peptide formation from different hexameric target sequences by the DnaE split-inteins from Synechocystis sp. and Nostoc punctiforme, revealing a strong impact of the target sequence and of the intein on the intracellular peptide concentration. Furthermore, we determined the crystal structures of their pre-splicing complexes, which allowed us to identify F-block Asp17 as crucial for the DnaE-mediated splicing reaction.

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“…The Npu C protein was purified using a high‐performance liquid chromatography (HPLC) system (Agilent Technologies) equipped with a Jupiter C18 column and obtained by lyophilization. The protein was quantified using a model 8453 The protein was quantified using a model 8453 UV/Vis spectrophotometer (Agilent Technologies) at 280 nm …”

Section: Methodsmentioning

confidence: 99%

Charge‐dependent modulation of specific and nonspecific protein‐metal ion interactions in nanoelectrospray ionization mass spectrometry

Zheng

Yuan

Hou

et al. 2019

Rapid Comm Mass Spectrometry

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Rationale Previous studies found that charge state could affect both specific and nonspecific binding of protein–metal ion interactions in nanoelectrospray ionization mass spectrometry (nESI‐MS). However, the two kinds of interactions have been studied individually in spite of the problem that they often coexist in the same system. Thus, it is necessary to study the effects of charge state on specific and nonspecific protein–metal ion interactions in one system to reveal more accurate binding state. Methods The HIV‐1 nucleocapsid protein (NCp7(31–55)) which can bind specifically and nonspecifically to Zn2+ served as the model to show the charge‐dependent protein–metal ion interactions. Hydrogen/deuterium exchange (HDX) and photodissociation (PD) were used to demonstrate that specific binding state was correlated with protein structure. In addition to NCp7(31–55), three other model proteins were used to investigate the reason for the charge‐dependent nonspecific binding. Results For specific binding, we proposed that protein ions with different charge states had different conformations. The HDX results showed that labile protons in the NCp7(31–55)–Zn complex were exchanged in a charge‐state‐dependent way. The PD experiments revealed differential fragment yields for different charge states. For nonspecific binding, higher charge states had more Zn2+ additions, but less SO42− additions. The effects of charge states on nonspecific binding levels were entirely the opposite for Zn2+ and SO42−. These results could reveal that the nonspecific binding was caused by electrostatic interaction. Conclusions For specific binding, NCp7(31–55) with lower charge states have folding and undenatured structures. The binding states of lower charge states can better reflect more native binding states. For nonspecific binding, when multiple metal ions adduct to proteins, the proteins have more net positive charges, which tend to generate higher charge ions during electrospray.

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Conserved residues that modulate proteintrans-splicing ofNpuDnaE split intein

Cited by 12 publications

References 49 publications

Design of a Split Intein with Exceptional Protein Splicing Activity

Design of a Split Intein with Exceptional Protein Splicing Activity

Mechanistic Insights into Cyclic Peptide Generation by DnaE Split‐Inteins through Quantitative and Structural Investigation

Charge‐dependent modulation of specific and nonspecific protein‐metal ion interactions in nanoelectrospray ionization mass spectrometry

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