pFind Studio: a computational solution for mass spectrometry-based proteomics
2017
PLOS ONE2017. Debelyy, MO et al.
Univ Fribourg, Div Biochem, Dept Biol, Fribourg, Switzerland.
ABSTRACT:Here we made an attempt to obtain partial structural information on the topology of multi-span integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).
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Biophysics Reports2017. Gong, Zhou et al.
CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, and National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, China
ABSTRACT:Chemical cross-linking coupled with mass spectroscopy (CXMS) is a powerful technique for investigating protein structures. CXMS has been mostly used to characterize the predominant structure for a protein, whereas cross-links incompatible with a unique structure of a protein or a protein complex are often discarded. We have recently shown that the so-called over-length cross-links actually contain protein dynamics information. We have thus established a method called DynaXL, which allow us to extract the information from the over-length cross-links and to visualize protein ensemble structures. In this protocol, we present the detailed procedure for using DynaXL, which comprises five steps. They are identification of highly confident cross-links, delineation of protein domains/subunits, ensemble rigid-body refinement, and final validation/assessment. The DynaXL method is generally applicable for analyzing the ensemble structures of multi-domain proteins and protein-protein complexes, and is freely available at www.tanglab.org/resources.
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Nature Microbiology2017. Said, N et al.
Free Univ Berlin, Lab Struct Biochem, Takustr 6, D-14195 Berlin, Germany.
ABSTRACT:lambda N-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein lambda N, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a lambda N-NusA-NusB-NusE-nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and lambda N, validated by crosslinking/mass spectrometry. Due to intrinsic disorder, lambda N can act as a multiprotein/RNA interaction hub, which, together with nut site RNA, arranges NusA, NusB and NusE into a triangular complex. This complex docks via the NusA N-terminal domain and the lambda N C-terminus next to the RNA exit channel on RNA polymerase. Based on the structures, comparative crosslinking analyses and structure-guided mutagenesis, we hypothesize that lambda N mounts a multipronged strategy to reprogram the transcriptional machinery, which may include (1) the lambda N C terminus clamping the RNA exit channel, thus stabilizing the DNA: RNA hybrid; (2) repositioning of NusA and RNAP elements, thus redirecting nascent RNA and sequestering the upstream branch of a terminator hairpin; and (3) hindering RNA engagement of termination factor rho and/or obstructing rho translocation on the transcript.
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eLife2017. Demo, G et al.
Univ Massachusetts, Sch Med, Dept Biochem & Mol Pharmacol, RNA Therapeut Inst, Worcester, MA 01655 USA.
ABSTRACT:In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30S. RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation.
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EMBO JOURNAL2017. Jakhanwal, S et al.
Max Planck Inst Biophys Chem, Dept Neurobiol, Gottingen, Germany.
ABSTRACT:Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18-1 as a possible acceptor complex for the R-SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18-1 remains tethered by the N-terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1-65) was able to bind to the syntaxin1: SNAP25: Munc18-1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R-SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly.
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Molecular Microbiology2017. Rebecca S. Bamert, Karl Lundquist, Hyea Hwang, Chaille T. Webb et al.
School of Physics, Georgia Institute of Technology, Atlanta, GA 30332, USA, Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
ABSTRACT:Theassemblyofproteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediatedbythe-barrelassemblymachinery(BAM). Two crucial elementsofthatmachineryarethecore BAM complex andthetranslocationandassemblymodule(TAM), with each containing a memberoftheOmp85 superfamilyofproteins: BamA intheBAM complex, TamA intheTAM. Here, we usedthesubstrateprotein FimD as a model to assesstheselectivityofsubstrateinteractionsfortheTAM relative to thoseoftheBAM complex. A peptide scan revealed that TamA and BamA bindthe-strandsofFimD, and do so selectively. Chemical cross-linking and molecular dynamics are consistent with this interaction taking place betweenthefirst and last strandoftheTamAbarreldomain, providingthefirst experimental evidenceofa lateral gate in TamA: astructuralelement implicated in membrane proteinassembly. We suggest thatthelateral gates in TamA and BamA provide different environmentsforsubstrates to engage, withthedifferences observed here beginning to address howtheTAM can be more effective thantheBAM complex inthefoldingofsomesubstrateproteins.
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Scientific Reports2017. Schmidt, C et al.
Univ Oxford, Dept Chem, Oxford, England.
ABSTRACT:ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of epsilon and delta subunits. Comparing cross-linking of naturally modified cATPase with the in vitro deacetylated enzyme revealed a major conformational change in the epsilon subunit in accord with extended and folded forms of the subunit. Locating modified residues within the catalytic head we found that phosphorylated and acetylated residues are primarily on alpha/beta and beta/alpha interfaces respectively. By aligning along different interfaces the higher abundance acetylated residues are proximal to the regulatory sites while the lower abundance phosphorylation sites are more densely populated at the catalytic sites. We propose that modifications in the catalytic head, together with the conformational change in subunit epsilon, work in synergy to fine-tune the enzyme during adverse conditions.
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Scientific Reports2017. Galgoczy, L et al.
Med Univ Innsbruck, Bioctr, Div Mol Biol, Innrain 80-82, A-6020 Innsbruck, Austria.
ABSTRACT:The recent global challenges to prevent and treat fungal infections strongly demand for the development of new antifungal strategies. The structurally very similar cysteine-rich antifungal proteins from ascomycetes provide a feasible basis for designing new antifungal molecules. The main structural elements responsible for folding, stability and antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and antifungal active protein. NFAP and its mutants carrying cysteine deletion (NFAP Delta C), hydrophobic core deletion (NFAP Delta h), and N-terminal amino acids exchanges (NFAP Delta N) were produced in Pichia pastoris. The recombinant NFAP showed the same features in structure, folding, stability and activity as the native protein. The data acquired with mass spectrometry, structural analyses and antifungal activity assays of NFAP and its mutants proved the importance of the disulphide bonding, the hydrophobic core and the correct N-terminus for folding, stability and full antifungal function. Our findings provide further support to the comprehensive understanding of the structure-function relationship in members of this protein group.
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Scientific Reports2017. Lundstrom, SL et al.
Karolinska Inst, Dept Med Biochem & Biophys, Div Physiol Chem 1, Stockholm, Sweden.
ABSTRACT:The human blood proteome is frequently assessed by protein abundance profiling using a combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS). In traditional sequence database search, many good-quality MS/MS data remain unassigned. Here we uncover the hidden part of the blood proteome via novel SpotLight approach. This method combines de novo MS/MS sequencing of enriched antibodies and co-extracted proteins with subsequent label-free quantification of new and known peptides in both enriched and unfractionated samples. In a pilot study on differentiating early stages of Alzheimer's disease (AD) from Dementia with Lewy Bodies (DLB), on peptide level the hidden proteome contributed almost as much information to patient stratification as the apparent proteome. Intriguingly, many of the new peptide sequences are attributable to antibody variable regions, and are potentially indicative of disease etiology. When the hidden and apparent proteomes are combined, the accuracy of differentiating AD (n = 97) and DLB (n = 47) increased from approximate to 85% to approximate to 95%. The low added burden of SpotLight proteome analysis makes it attractive for use in clinical settings.
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Journal of Proteome Research2017. Blank-Landeshammer, B et al.
ISAS eV, Leibniz Inst Analyt Wissensch, D-44139 Dortmund, Germany.
ABSTRACT:Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms. Results were validated using state-of-the art database search algorithms as well specifically synthesized reference peptides. Thus, we could increase the number of PSMs meeting a stringent FDR of 5% more than 3-fold compared to the single best de novo sequencing algorithm alone, accounting for an average of 11 120 PSMs (combined) instead of 3476 PSMs (alone) in triplicate 2 h LC-MS runs of tryptic HeLa digestion.
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