In Cellulo Protein Semi-Synthesis from Endogenous and Exogenous Fragments Using the Ultra-Fast Split Gp41-1 Intein

Protein semi-synthesis inside live cells from exogenous and endogenous parts offers unique possibilities for studying proteins in their native context. Split-intein-mediated protein trans-splicing is predestined for such endeavors and has seen some successes, but a much larger variety of established split inteins and associated protocols is urgently needed. We characterized the association and splicing parameters of the Gp41-1 split intein, which favorably revealed a nanomolar affinity between the intein fragments combined with the exceptionally fast splicing rate. Following bead-loading of a chemically modified intein fragment precursor into live mammalian cells, we fluorescently labeled target proteins on their N- and C-termini with short peptide tags, thus ensuring minimal perturbation of their structure and function. In combination with a nuclear-entrapment strategy to minimize cytosolic fluorescence background, we applied our technique for super-resolution imaging and single-particle tracking of the outer mitochondrial protein Tom20 in HeLa cells.     

Generation of a Genetically Encoded,Photoactivatable Intein for the Controlled Production of Cyclic Peptides

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split‐intein‐mediated SICLOPPS technology provides a generic access to fully genetically encoded head‐to‐tail cyclized peptides and large libraries thereof (SICLOPPS=split‐intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light‐dependent intein using the photocaged tyrosine derivative ortho‐nitrobenzyltyrosine incorporated at an internal, non‐catalytic position is now reported. Stable intein precursors were purified from the E. coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.     

Generation of a Genetically Encoded,Photoactivatable Intein for the Controlled Production of Cyclic Peptides

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split‐intein‐mediated SICLOPPS technology provides a generic access to fully genetically encoded head‐to‐tail cyclized peptides and large libraries thereof (SICLOPPS=split‐intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light‐dependent intein using the photocaged tyrosine derivative ortho‐nitrobenzyltyrosine incorporated at an internal, non‐catalytic position is now reported. Stable intein precursors were purified from the E. coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.     

An Atypical Naturally Split Intein Engineered for Highly Efficient Protein Labeling

Protein trans‐splicing catalyzed by split inteins is a powerful technique for assembling a polypeptide backbone from two separate parts. However, split inteins with robust efficiencies and short fragments suitable for peptide synthesis are rare and have mostly been artificially created. The novel split intein AceL‐TerL was identified from metagenomic data and characterized. It represents the first naturally occurring, atypically split intein. The N‐terminal fragment of only 25 amino acids is the shortest natural intein fragment to date and was easily amenable to chemical synthesis with a fluorescent label. Optimal protein trans‐splicing activity was observed at low temperatures. Further improved mutants were selected by directed protein evolution. The engineered intein variants with up to 50‐fold increased rates showed unprecedented efficiency in chemically labeling of a diverse set of proteins. These inteins should prove valuable tools for protein semi‐synthesis and other intein‐related biotechnological applications.     

Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo

Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.     

Click‐Tag and Amine‐Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein

Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is transferred to the protein of interest by protein trans‐splicing. For the amine‐tag, a recombinant intein fragment free of lysine residues was generated such that the amine group of the N terminus could be selectively modified with regular amine‐reactive reagents. Thus, standard bioconjugation procedures without any chemical synthesis could be applied without modification of lysines in the protein of interest. For the click‐tag, protein trans‐splicing was combined with unnatural amino acid mutagenesis and subsequent bioorthogonal side chain modification, as demonstrated for click chemistry using p‐azidophenylalanine. By the two‐step strategy, exposure of the protein of interest to the copper catalyst was avoided.     

Intramolecular disulfide bond between catalytic cysteines in an intein precursor

Protein splicing is a self-catalyzed and spontaneous post-translational process in which inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic Pyrococcus abyssi PolII intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography-mass spectrometry (LC-MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that inteins may act as switches to control extein function.     

Design of an intein that can be inhibited with a small molecule ligand

Protein splicing is a process in which an intervening sequence, the intein, catalyzes its own excision out of a larger polypeptide precursor by joining the flanking sequences, the exteins, with a native peptide bond. Inteins are almost completely promiscuous toward the nature of their extein sequences and can be inserted into virtually any host protein. The intein-mediated formation of a peptide bond between two polypeptides offers great potential to modulate protein structure and, hence, protein function on the post-translational level. In this work, we report the design of an intein that can be inhibited by the addition of a specific small molecule ligand. Our design strategy involved the generation of a trans-splicing intein, in which the intein domain is split into two-halves that are located on two separate polypeptides, each joined with the respective N- or C-terminal extein. To turn these fragments into an active intein with an incorporated "off" switch, each was fused at its newly created terminus with the F36M mutant of FKBP12, referred to as the FM domain. The F36M substitution was reported to effect a homodimerization of the usually monomeric FKBP12 protein; however, addition of the small molecule ligand, rapamycin, or synthetic derivatives thereof leads to a dissociation of the dimer. This phenomenon was exploited by first reconstituting the active intein on the basis of FM domain dimerization. Second, addition of the small molecule ligand prevented formation of the active intein complex and inhibited protein trans-splicing. This intein exhibited unexpected kinetic properties and provides a new and potentially very general means to control protein function on the post-translational level.     

pK(a) coupling at the intein active site: implications for the coordination mechanism of protein splicing with a conserved aspartate

Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pK(a) of 6.1, two units above the normal pK(a) of aspartate. The elevated pK(a) of D422 is coupled to the depressed pK(a) of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pK(a) to normal, while a D422G mutation increases the C1 pK(a) from 7.5 to 8.5. The pK(a) coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N-S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing.     

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.     

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated ¹⁵N-labeling of the C-terminal amide group of a single domain antibody (V_HH).     

Site specific biotinylation of the human aldo/keto reductase AKR1A1 for immobilization

New strategies for the specific monolabelling of enzymes play a key role in the development of artificial proteins. Especially for the emerging research field of nanobiotechnology and bioelectronics artificial monofunctionalized redoxproteins are of great interest. The human AKR1A1, an enzyme of the aldo/keto reductase superfamily, has been chosen as subject for the synthesis of an artificially mono biotinylated redoxprotein in order to selectively immobilize this enzyme for bioelectronic applications. To produce monofunctionalized enzyme we applied the strategy of Expressed Protein Ligation (EPL) in combination with solid phase peptide synthesis (SPPS). Accordingly, we used the IMPACT^®-system and cloned the aldo/keto-reductase as fusion protein with an additional intein/chitin binding domain. Through intein mediated splicing we could produce the C-terminal thioester of the aldo/keto-reductase, which maintained its biological activity. Then, the thioester was coupled to Cys-Lys(Ahx-Ahx-biotin)-amide by Native Chemical Ligation, which led to mono-biotinylated protein. The enzyme activity was proven to be intact as shown by various kinetic investigations. Immobilization was performed on avidin coated silica microspheres. Accordingly, for the first time selectively modified AKR1A1 has been immobilized.     

Mechanism of C-terminal intein cleavage in protein splicing from QM/MM molecular dynamics simulations

Protein splicing is a post-translational process in which a biologically inactive protein is activated by the release of a segment denoted as an intein. The process involves four steps. In the third, the scission of the intein takes place after the cyclization of the last amino acid of the segment, an asparagine. Little is known about the chemical reaction necessary for this cyclization. Experiments demonstrate that two histidines (the penultimate amino acid of the intein, and a histidine located 10 amino acids upstream) are relevant in the cyclization of the asparagine. We have investigated the mechanism and determinants of reaction in the GyrA intein focusing on the requirements for asparagine activation for its cyclization. First, the influence that the protonation states of these two histidines have on the orientation of the asparagine side chain is investigated by means of molecular dynamics simulation. Molecular dynamics simulations using the CHARMM27 force field were carried out on the three possible protonation states for each of these two histidines. The results indicate that the only protonation state in which the conformation of the system is suitable for cyclization is when the penultimate histidine is fully protonated (positively charged), and the upstream histidine is in the His(ε) neutral tautomeric form. The free energy profile for the reaction in which the asparagine is activated by a proton transfer to the upstream histidine is presented, computed by hybrid quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics at the SCCDFTB/CHARMM27 level of theory. The calculated free energy barrier for the reaction is 19.0 kcal mol(-1). B3LYP/6-31+G(d) QM/MM single-point calculations give a qualitatively a similar energy profile, although with somewhat higher energy barriers, in good agreement with the value derived from experiment of 25 kcal mol(-1) at 60 °C. QM/MM molecular dynamics simulations of the reactant, activated reactant and intermediate states highlight the importance of the Arg181-Val182-Asp183 segment in catalysing the reaction. Overall, the results indicate that nucleophilic activation of the asparagine for its cyclization by the upstream histidine acting as the base is a plausible mechanism for the C-terminal cleavage in protein splicing.     

RNA-Targeting Splicing Modifiers: Drug Development and Screening Assays

RNA splicing is an essential step in producing mature messenger RNA (mRNA) and other RNA species. Harnessing RNA splicing modifiers as a new pharmacological modality is promising for the treatment of diseases caused by aberrant splicing. This drug modality can be used for infectious diseases by disrupting the splicing of essential pathogenic genes. Several antisense oligonucleotide splicing modifiers were approved by the U.S. Food and Drug Administration (FDA) for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD). Recently, a small-molecule splicing modifier, risdiplam, was also approved for the treatment of SMA, highlighting small molecules as important warheads in the arsenal for regulating RNA splicing. The cellular targets of these approved drugs are all mRNA precursors (pre-mRNAs) in human cells. The development of novel RNA-targeting splicing modifiers can not only expand the scope of drug targets to include many previously considered “undruggable” genes but also enrich the chemical-genetic toolbox for basic biomedical research. In this review, we summarized known splicing modifiers, screening methods for novel splicing modifiers, and the chemical space occupied by the small-molecule splicing modifiers.     

The Mechanism of Cholesterol Modification of Hedgehog Ligand

Hedgehog (Hh) proteins are important components of signal transduction pathways involved in animal development, and their defects are implicated in carcinogenesis. Their N-terminal domain (HhN) acts as a signaling ligand, and their C-terminal domain (HhC) performs an autocatalytic function of cleaving itself away, while adding a cholesterol moiety to HhN. HhC has two sub-domains: a hedgehog/intein (hint) domain that primarily performs the autocatalytic activity, and a sterol-recognition region (SRR) that binds to cholesterol and properly positions it with respect to HhN. The three-dimensional details of this autocatalytic mechanism remain unknown, as does the structure of the precursor Hh protein. In this study, a complete cholesterol-bound precursor form of the drosophila Hh precursor is modeled using known crystal structures of HhN and the hint domain, and a hypothesized similarity of SRR to an unrelated but similar-sized cholesterol binding protein. The restrained geometries and topology switching (RGATS) strategy is then used to predict atomic-detail pathways for the full autocatalytic reaction starting from the precursor and ending in a cholesterol-linked HhN domain and a cleaved HhC domain. The RGATS explicit solvent simulations indicate the roles of individual HhC residues in facilitating the reaction, which can be confirmed through mutational experiments. These simulations also provide plausible structural models for the N/S acyl transfer intermediate and the product states of this reaction. This study thus provides a good framework for future computational and experimental studies to develop a full structural and dynamic understanding of Hh autoprocessing. © 2019 Wiley Periodicals, Inc.     

钯配合物对结核杆菌RecA intein蛋白质剪接的抑制作用

研究了5种钯配合物Pd(bipy)Cl₂, Pd(phen)Cl₂, [Pd(dien)Cl]Cl, trans-Pd(NH₃)₂Cl₂和cis-Pd(NH₃)₂Cl₂对结核杆菌RecA intein蛋白质剪接的抑制作用. 结果表明, trans-Pd(NH₃)₂Cl₂的抑制活性最好, IC₅₀=3.3×10^-5 mol/L. 钯配合物通过与intein的第一个氨基酸(半胱氨酸)配位, 从而抑制蛋白质的剪接活性. 荧光猝灭的动力学数据表明, 配体的大小会明显影响钯配合物与intein的相互作用, 配体越大, 作用越慢.     

Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α‐Synuclein‐Induced Cytotoxicity in Baker′s Yeast

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans‐splicing in combination with a highly efficient split‐intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI‐I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α‐synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI‐I in a ratio of 1:5×10⁴. This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide‐based libraries in eukaryotic cells.     

Effect of the linear siloxane chain in cyclic silsesquioxane (CSSQ) on the mechanical/electrical property of the thin films

Several kinds of cyclic silsesquioxane (CSSQ) precursors containing linear siloxane chain were prepared to improve both the mechanical properties of their thin films and the compatibility with heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin (tCD) as a porogen. The precursors were synthesized using a hydrolysis/condensation reaction with 2,4,6,8-tetramethyl-2,4,6,8-tetra (trimethoxysilylethyl) cyclotetrasiloxane (cyclic monomer) and three kinds of linear siloxane monomers. As the linear siloxane chain length increases in the CSSQ precursors, the compatibility between the CSSQ precursor and tCD molecules improved due to the chain flexibility of the precursor. Moreover, the mechanical strength of the CSSQ precursor (4ST37) containing linear tetrasiloxane was the best among the prepared precursors. The enhancement of mechanical property might also be attributed to the content of Si-OH groups as well as the chain flexibility, which could help the crosslinking reaction of Si-OH groups in the film curing process.     

The intein of the Thermoplasma A-ATPase A subunit: Structure, evolution and expression in E. coli

Background  

Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation.