66 research outputs found
Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site
Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.Peer reviewe
The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from cis-splicing inteins
Abstract Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 Ã…-resolution crystal structure of a fused variant from the naturally split gp41-1 intein, previously identified from environmental metagenomic sequence data. The structure of the 125-residue gp41-1 intein revealed a compact pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N- and C-terminal intein fragments that are common among naturally occurring split inteins. We successfully created orthogonal split inteins by engineering a similar charge network into the same region of a cis-splicing intein. This strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins.Peer reviewe
Validated UPLC-MS/MS methods to quantitate free and conjugated Alternaria toxins in commercially available tomato products and fruit and vegetable juices in Belgium
Ultraperformance liquid chromatography tandem mass spectrometry and Quick, Easy, Cheap, Effective, Rugged, and Safe based analytical methodologies to quantitate both free (alternariol (1), alternariol monomethyl ether (2), tenuazonic acid (3), tentoxin (4), altenuene (5), altertoxin-I (6)) and conjugated (sulfates and glucosides of 1 and 2) Alternaria toxins in fruit and vegetable juices and tomato products were developed and validated. Acceptable limits of quantitation (0.7-5.7 mu g/kg), repeatability (RSDr < 15.7%), reproducibility (RSDR < 17.9%), and apparent recovery (87.0-110.6%) were obtained for all analytes in all matrices investigated. 129 commercial foodstuffs were analyzed, and 3 was detected in 100% of tomato product samples (<LOQ to 333 mu g/kg), while 1, 2, 4, and 5 were also frequently detected (21-86%, <LOQ to 62 mu g/kg). Moreover, low levels (<LOQ to 9.9 mu g/kg) of modified Alternaria toxins (sulfates of 1 and 2) were repeatedly detected. A deterministic dietary exposure assessment revealed the possible risk for human health related to the presence of 1 and 2 in tomato based foodstuffs, whereas 3 is unlikely to be of human health concern
NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein
The growing understanding of partially unfolded proteins increasingly points to their biological relevance in allosteric regulation, complex formation, and protein design. However, the structural characterization of disordered proteins remains challenging. NMR methods can access both the dynamics and structures of such proteins, yet suffering from a high degeneracy of NMR signals. Here, we overcame this bottleneck utilizing a salt-inducible split intein to produce segmentally isotope-labeled samples with the native sequence, including the ligation junction. With this technique, we investigated the NMR structure and conformational dynamics of TonB from Helicobacter pylori in the presence of a proline-rich low complexity region. Spin relaxation experiments suggest that the several nano-second time scale dynamics of the C-terminal domain (CTD) is almost independent of the faster pico-to-nanosecond dynamics of the low complexity central region (LCCR). Our results demonstrate the utility of segmental isotopic labeling for proteins with heterogenous dynamics such as TonB and could advance NMR studies of other partially unfolded proteins.Peer reviewe
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Design and Development of Fluorescent Vemurafenib Analogs for In Vivo Imaging
Herein we describe fluorescent derivatives of vemurafenib to probe therapeutic BRAF inhibition in live cells and in vivo. The compounds were evaluated and compared by determining target binding, inhibition of mutant BRAF melanoma cell lines and live cell imaging. We show that vemurafenib-BODIPY is a superior imaging drug to visualize the targets of vemurafenib in live cells and in vivo in non-resistant and resistant melanoma tumors
Tracking emerging mycotoxins in food: development of an LC-MS/MS method for free and modified Alternaria toxins
Mycotoxins produced by Alternaria fungi are ubiquitous food contaminants, but analytical methods for generating comprehensive exposure data are rare. We describe the development of an LC-MS/MS method covering 17 toxins for investigating the natural occurrence of free and modified Alternaria toxins in tomato sauce, sunflower seed oil, and wheat flour. Target analytes included alternariol (AOH), AOH-3-glucoside, AOH-9-glucoside, AOH-3-sulfate, alternariol monomethyl ether (AME), AME-3-glucoside, AME-3-sulfate, altenuene, isoaltenuene, tenuazonic acid (TeA), tentoxin (TEN), altertoxin I and II, alterperylenol, stemphyltoxin III, altenusin, and altenuic acid III. Extensive optimization resulted in a time- and cost-effective sample preparation protocol and a chromatographic baseline separation of included isomers. Overall, adequate limits of detection (0.03–9 ng/g) and quantitation (0.6–18 ng/g), intermediate precision (9–44%), and relative recovery values (75–100%) were achieved. However, stemphyltoxin III, AOH-3-sulfate, AME-3-sulfate, altenusin, and altenuic acid III showed recoveries in wheat flour below 70%, while their performance was stable and reproducible. Our pilot study with samples from the Austrian retail market demonstrated that tomato sauces (n = 12) contained AOH, AME, TeA, and TEN in concentrations up to 20, 4, 322, and 0.6 ng/g, while sunflower seed oil (n = 7) and wheat flour samples (n = 9) were contaminated at comparatively lower levels. Interestingly and of relevance for risk assessment, AOH-9-glucoside, discovered for the first time in naturally contaminated food items, and AME-3-sulfate were found in concentrations similar to their parent toxins. In conclusion, the established multi-analyte method proved to be fit for purpose for generating comprehensive Alternaria toxin occurrence data in different food matrices
The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms
Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues
Natural occurrence, exposure assessment & risk characterization of Alternaria mycotoxins in apple by‑products in Argentina
Data on the natural occurrence of Alternaria mycotoxins in apple by-products is lacking in Argentina and the risk of exposure to these mycotoxins has not been characterized before. The levels of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN), altertoxin-I (ATX-I), altertoxin-II (ATX-II), alternariol 3-sulfate (AOH-3-S), alternariol 3-glucoside (AOH-3-G), alternariol monomethyl ether 3-sulfate (AME-3-S), and alternariol monomethyl ether 3-glucoside (AME-3-G) were determined in clarified and cloudy apple juices, marmalades, and apple-based infant food from the Argentinean market, and the risk of exposure was characterized. Detectable levels of AME, TEN, TeA, AME-3-S and AOH-3-G were found in clarified juices, while the same mycotoxins plus AOH were found in cloudy apple juices in higher concentrations. AME, TEN, TeA and AOH-3G were detected in marmalades, and AOH, AME, TEN and TeA in apple infant food. Probabilistic exposure assessment and risk characterization were carried out for children between 6 months and 5 years old in Argentina. The highest risk of exposure affected children between 6 and 23 months from the consumption of apple infant food and mainly associated with the alternariols. Better control strategies to prevent the incorporation of Alternaria mouldy core into the process line and the establishment of legislation for Alternaria mycotoxins are needed in Argentina
Vernacular museum: communal bonding and ritual memory transfer among displaced communities
Eclectically curated and largely ignored by the mainstream museum sector, vernacular museums sit at the interstices between the nostalgic and the future-oriented, the private and the public, the personal and the communal. Eluding the danger of becoming trivialised or commercialised, they serve as powerful conduits of memory, which strengthen communal bonds in the face of the ‘flattening’ effects of globalisation. The museum this paper deals with, a vernacular museum in Vanjärvi in southern Finland, differs from the dominant type of the house museum, which celebrates masculinity and social elites. Rather, it aligns itself with the small amateur museums of everyday life called by Angela Jannelli Wild Museums (2012), by analogy with Lévi-Strauss’ concept of ‘pensée sauvage’. The paper argues that, despite the present-day flurry of technologies of remembering and lavishly funded memory institutions, there is no doubt that the seemingly ‘ephemeral’ institutions such as the vernacular museum, dependent so much on performance, oral storytelling, living bodies and intimate interaction, nevertheless play an important role in maintaining and invigorating memory communities
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