Exogenous carbonaceous contaminants coming from sediments significantly bias the radiocarbon date of collagen samples extracted from archaeological bone and teeth. In this study, a new approach ...combining pyrolysis, comprehensive gas chromatography and mass spectrometry (Py-GC × GC/MS) was proposed to ensure their removal during the demineralization and bone collagen extraction. This approach permitted to identify hydrocarbon contaminants for archaeological samples from the Neolithic period, in 30–40 μg of collagen. The use of 2D GC improved importantly the separation, selectivity and resolution compared to 1D GC thus permitting to detect organic contaminants within the complex chromatograms issued from collagen pyrolysis. Moreover, efficiency of the extraction steps in collagen sample preparation for radiocarbon dating (acid and alkali treatments, filtration steps) could be evaluated for four different protocols on the basis of organic contaminant removal. Radiocarbon dating of the extracted collagen of four of the tested protocols corroborated the results of the Py GC × GC/MS data. This approach opens new perspectives for the use of comprehensive gas chromatography in the domain of archaeological sciences.
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•Soil contaminants in archaeological collagen may be detected upstream.•Collagen extraction protocols are differently effective for removing contaminants.•Py-GCxGC/MS is efficient to assess contaminant persistence and collagen preservation.
Bone remains of small vertebrate fossils provide valuable information for paleoenvironmental and paleoclimatic reconstructions. However, direct radiocarbon dating of small vertebrates remains ...challenging as the extraction of sufficient good quality collagen is required. The efficiency of eight collagen extraction protocols was tested on seven samples, representative of different ages and burial environments, including both macro and small vertebrate taxa. First, the samples were prescreened using attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) to quantify collagen content in archaeological bones, revealing that one should be discarded for 14C dating. Then, the quantity of protein extracted (yield) and collagen integrity were checked using conventional elemental analysis. The results show that one protocol was not able to accurately extract collagen from the samples. A soft HCl-based protocol seems more appropriate for the pretreatment of archaeological small mammal bones, whereas a harsher protocol might be more efficient to extract a higher amount of collagen from large mammals as well as amphibian bones. The influence of the tested protocols on carbon and nitrogen isotope values was also investigated. The results showed that isotopic variability, when existing, is related to the interindividual differences rather than the different protocols.
Lasso peptides constitute a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the ...C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Tandem mass spectrometry using collision induced dissociation (CID) and electron capture dissociation (ECD) have shown previously different fragmentation patterns for capistruin, microcin J25 and their corresponding branched-cyclic forms in which the C-terminal tail is unthreaded. In order to develop general rules that unambiguously discriminate the lasso and branched-cyclic topologies, this report presents experimental evidence for a set of twenty-one lasso peptides analyzed by CID and electron transfer dissociation (ETD). CID experiments on lasso peptides specifically yielded mechanically interlocked species with associated b
and y
fragments. For class II lasso peptides, these lasso-specific fragments were observed only for peptides in which the loop, located above the macrolactam ring, was strictly longer than four amino acid residues. For class I and III lasso peptides, part of the C-terminal tail remains covalently linked to the macrolactam ring by disulfide bonds; associated b
and y
fragments therefore do not clearly constitute a signature of the lasso topology. ETD experiments of lasso peptides showed a significant increase of hydrogen migration events in the loop region when compared to their branched-cyclic topoisomers, leading to the formation of specific c
˙/z'
fragments for all lasso peptides, regardless of their class and loop size. Our experiments enabled us to establish general rules for obtaining structural details from CID and ETD fragmentation patterns, obviating the need for structure determination by NMR or X-ray crystallography.
Because hard tissues can be radiocarbon dated, they are key to establishing the archaeological chronologies, palaeoenvironmental reconstructions and historical-biogeographical processes of the last ...50,000 years. The advent of accelerator mass spectrometers (AMS) has revolutionized the field of archaeology but routine AMS dating still requires 60-200 mg of bone, which far exceeds that of small vertebrates or remains which hold a patrimonial value (e.g. hominid remains or worked bone artefacts). Here, we present the first radiocarbon dates obtained from minute amounts of bone (3-60 mg) using a MIni CArbon DAting System (MICADAS). An optimized protocol allowed us to extract enough material to produce between 0.2 and 1.0 mg of carbon for graphite targets. Our approach was tested on known-age samples dating back to 40,000 BP, and served as proof of concept. The method was then applied to two archaeological sites where reliable dates were obtained from the single bones of small mammals. These results open the way for the routine dating of small or key bone samples.
Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ...ATP-binding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-Å resolution, which is to the authors’ knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3–6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outward-occluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters.
Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that ...utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.
Synopsis
Bacteria employ dedicated ABC transporters to secrete antibacterial peptides. X‐ray structure analysis shows that the antibacterial lasso peptide transporter McjD, in contrast to other ABC transporters, lacks a stable open cavity conformation, thus preventing the influx of the exported toxic peptide.
The structure of the ABC transporter McjD was determined in two distinct conformations, apo and nucleotide‐bound.
Both conformations show an occluded cavity at both sides of the membrane.
Pulsed electron‐electron double resonance (PELDOR) measurements in the presence of the antibacterial peptide MccJ25 did not identify a stable open‐conformation.
Cross‐linking studies in proteoliposomes show that the cavity opens transiently to release the bound substrate.
Transient opening of the antibacterial peptide transporter McjD prevents the influx of its toxic substrate MccJ25 and distinguishes it from other multi‐drug transporters.
Statistical heterospectroscopy (SHY) is a new statistical paradigm for the coanalysis of multispectroscopic data sets acquired on multiple samples. This method operates through the analysis of the ...intrinsic covariance between signal intensities in the same and related molecules measured by different techniques across cohorts of samples. The potential of SHY is illustrated using both 600-MHz 1H NMR and UPLC-TOFMS data obtained from control rat urine samples (n = 54) and from a corresponding hydrazine-treated group (n = 58). We show that direct cross-correlation of spectral parameters, viz. chemical shifts from NMR and m/z data from MS, is readily achievable for a variety of metabolites, which leads to improved efficiency of molecular biomarker identification. In addition to structure, higher level biological information can be obtained on metabolic pathway activity and connectivities by examination of different levels of the NMR to MS correlation and anticorrelation matrixes. The SHY approach is of general applicability to complex mixture analysis, if two or more independent spectroscopic data sets are available for any sample cohort. Biological applications of SHY as demonstrated here show promise as a new systems biology tool for biomarker recovery.
Amyloid deposits within the cerebral tissue constitute a characteristic lesion associated with Alzheimer disease. They mainly consist of the amyloid peptide Aβ and display an abnormal content in Zn2+ ...ions, together with many truncated, isomerized, and racemized forms of Aβ. The region 1-16 of Aβ can be considered the minimal zinc-binding domain and contains two aspartates subject to protein aging. The influence of zinc binding and protein aging related modifications on the conformation of this region of Aβ is of importance given the potentiality of this domain to constitute a therapeutic target, especially for immunization approaches. In this study, we determined from NMR data the solution structure of the Aβ-(1-16)-Zn2+ complex in aqueous solution at pH 6.5. The residues His6, His13, and His14 and the Glu11 carboxylate were identified as ligands that tetrahedrally coordinate the Zn(II) cation. In vitro aging experiments on Aβ-(1-16) led to the formation of truncated and isomerized species. The major isomer generated, Aβ-(1-16)-l-iso-Asp7, displayed a local conformational change in the His6-Ser8 region but kept a zinc binding propensity via a coordination mode involving l-iso-Asp7. These results are discussed here with regard to Aβ fibrillogenesis and the potentiality of the region 1-16 of Aβ to be used as a therapeutic target.
Lasso peptides constitute a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the ...C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Tandem mass spectrometry using collision induced dissociation (CID) and electron capture dissociation (ECD) have shown previously different fragmentation patterns for capistruin, microcin J25 and their corresponding branched-cyclic forms in which the C-terminal tail is unthreaded. In order to develop general rules that unambiguously discriminate the lasso and branched-cyclic topologies, this report presents experimental evidence for a set of twenty-one lasso peptides analyzed by CID and electron transfer dissociation (ETD). CID experiments on lasso peptides specifically yielded mechanically interlocked species with associated
b
i
and
y
j
fragments. For class II lasso peptides, these lasso-specific fragments were observed only for peptides in which the loop, located above the macrolactam ring, was strictly longer than four amino acid residues. For class I and III lasso peptides, part of the C-terminal tail remains covalently linked to the macrolactam ring by disulfide bonds; associated
b
i
and
y
j
fragments therefore do not clearly constitute a signature of the lasso topology. ETD experiments of lasso peptides showed a significant increase of hydrogen migration events in the loop region when compared to their branched-cyclic topoisomers, leading to the formation of specific
c
i
&z.rad;/
z
′
j
fragments for all lasso peptides, regardless of their class and loop size. Our experiments enabled us to establish general rules for obtaining structural details from CID and ETD fragmentation patterns, obviating the need for structure determination by NMR or X-ray crystallography.
Lasso peptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring.
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