Publications
243 results found
McKenna S, Aylward F, Miliara X, et al., 2023, The protease associated (PA) domain in ScpA from Streptococcus pyogenes plays a role in substrate recruitment, BBA: Proteins and Proteomics, Vol: 1871, Pages: 1-11, ISSN: 1570-9639
Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage.
Jønsson R, Björling A, Midtgaard SR, et al., 2023, Aggregative adherence fimbriae form compact structures as seen by SAXS., Sci Rep, Vol: 13
Bacterial colonization is mediated by fimbriae, which are thin hair-like appendages dispersed from the bacterial surface. The aggregative adherence fimbriae from enteroaggregative E. coli are secreted through the outer membrane and consist of polymerized minor and major pilin subunits. Currently, the understanding of the structural morphology and the role of the minor pilin subunit in the polymerized fimbriae are limited. In this study we use small-angle X-ray scattering to reveal the structural morphology of purified fimbriae in solution. We show that the aggregative fimbriae are compact arrangements of subunit proteins Agg5A + Agg3B which are assembled pairwise on a flexible string rather than extended in relatively straight filaments. Absence of the minor subunit leads to less compact fimbriae, but did not affect the length. The study provides novel insights into the structural morphology and assembly of the aggregative adherence fimbriae. Our study suggests that the minor subunit is not located at the tip of the fimbriae as previously speculated but has a higher importance for the assembled fimbriae by affecting the global structure.
Karamanos TK, Matthews S, 2023, Biomolecular NMR in the AI-assisted structural biology era: Old tricks and new opportunities., Biochim Biophys Acta Proteins Proteom
Over the last 40 years nuclear magnetic resonance (NMR) spectroscopy has established itself as one of the most versatile techniques for the characterization of biomolecules, especially proteins. Given the molecular size limitations of NMR together with recent advances in cryo-electron microscopy and artificial intelligence-assisted protein structure prediction, the bright future of NMR in structural biology has been put into question. In this mini review we argue the contrary. We discuss the unique opportunities solution NMR offers to the protein chemist that distinguish it from all other experimental or computational methods, and how it can benefit from machine learning.
Vadas O, Pacheco NDS, Chao K, et al., 2023, Structural and regulatory insights into the glideosome- associated connector from Toxoplasma gondii, eLife, Vol: 12, ISSN: 2050-084X
The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells, and cross biological barriers. The glideosome-associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the solution properties together with membrane and F-actin binding interfaces suggests that GAC adopts several conformations from closed to open and extended. A multi-conformational model for assembly and regulation of GAC within the glideosome is proposed.
Rasmussen HO, Kumar A, Shin B, et al., 2023, FapA is an Intrinsically Disordered Chaperone for<i> Pseudomonas</i> Functional Amyloid FapC, JOURNAL OF MOLECULAR BIOLOGY, Vol: 435, ISSN: 0022-2836
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- Citations: 4
Miliara X, Tatsuta T, Eiyama A, et al., 2023, An intermolecular hydrogen bonded network in the PRELID-TRIAP protein family plays a role in lipid sensing, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, Vol: 1871, ISSN: 1570-9639
Meisl G, Xu CK, Taylor JD, et al., 2022, Uncovering the universality of self-replication in protein aggregation and its link to disease, Science Advances, Vol: 8, Pages: 1-11, ISSN: 2375-2548
Fibrillar protein aggregates are a hallmark of a range of human disorders, from prion diseases to dementias, but are also encountered in several functional contexts. Yet, the fundamental links between protein assembly mechanisms and their functional or pathological roles have remained elusive. Here, we analyze the aggregation kinetics of a large set of proteins that self-assemble by a nucleated-growth mechanism, from those associated with disease, over those whose aggregates fulfill functional roles in biology, to those that aggregate only under artificial conditions. We find that, essentially, all such systems, regardless of their biological role, are capable of self-replication. However, for aggregates that have evolved to fulfill a structural role, the rate of self-replication is too low to be significant on the biologically relevant time scale. By contrast, all disease-related proteins are able to self-replicate quickly compared to the time scale of the associated disease. Our findings establish the ubiquity of self-replication and point to its potential importance across aggregation-related disorders.
Zhang K, Li S, Wang Y, et al., 2022, Bacteriophage protein PEIP is a potent<i> Bacillus</i><i> subtilis</i> enolase inhibitor, CELL REPORTS, Vol: 40, ISSN: 2211-1247
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- Citations: 3
Kelsall IR, McCrory EH, Xu Y, et al., 2022, HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation, EMBO JOURNAL, Vol: 41, ISSN: 0261-4189
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- Citations: 17
McKenna S, Huse KK, Giblin S, et al., 2022, The role of streptococcal cell-envelope proteases in bacterial evasion of the innate immune system, Journal of Innate Immunity, Vol: 14, Pages: 69-88, ISSN: 1662-811X
Bacteria possess the ability to evolve varied and ingenious strategies to outwit the host immune system, instigating an evolutionary arms race. Proteases are amongst the many weapons employed by bacteria, which specifically cleave and neutralize key signalling molecules required for a coordinated immune response. In this article, we focus on a family of S8 subtilisin-like serine proteases expressed as cell-envelope proteases (CEPs) by group A and group B streptococci. Two of these proteases known as Streptococcus pyogenes CEP (SpyCEP) and C5a peptidase cleave the chemokine CXCL8 and the complement fragment C5a, respectively. Both CXCL8 and C5a are potent neutrophil-recruiting chemokines, and by neutralizing their activity, streptococci evade a key defence mechanism of innate immunity. We review the mechanisms by which CXCL8 and C5a recruit neutrophils and the characterization of SpyCEP and C5a peptidase, including both in vitro and in vivo studies. Recently described structural insights into the function of this CEP family are also discussed. We conclude by examining the progress of prototypic vaccines incorporating SpyCEP and C5a peptidase in their preparation. Since streptococci-producing SpyCEP and C5a peptidase are responsible for a considerable global disease burden, targeting these proteases by vaccination strategies or by small-molecule antagonists should provide protection from and promote the resolution of streptococcal infections.
Zhang P, Zhao X, Wang Y, et al., 2022, Bacteriophage protein Gp46 is a cross-species inhibitor of nucleoid-associated HU proteins, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 119, ISSN: 0027-8424
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- Citations: 7
Matthews S, 2022, Secondary structure and X-ray crystallographic analysis of the Glideosome-Associated Connector (GAC) from Toxoplasma gondii, Crystals, Vol: 12, Pages: 1-6, ISSN: 2073-4352
A model for parasitic motility has been proposed in which parasite filamentous actin (F-actin) is attached to surface adhesins by a large component of the glideosome, known as the glideosome-associated connector protein (GAC). This large 286 kDa protein interacts at the cytoplasmic face of the plasma membrane with the phosphatidic acid-enriched inner leaflet and cytosolic tails of surface adhesins to connect them to the parasite actomyosin system. GAC is observed initially to the conoid at the apical pole and re-localised with the glideosome to the basal pole in gliding parasite. GAC presumably functions in force transmission to surface adhesins in the plasma membrane and not in force generation. Proper connection between F-actin and the adhesins is as important for motility and invasion as motor operation itself. This notion highlights the need for new structural information on GAC interactions, which has eluded the field since its discovery. We have obtained crystals that diffracted to 2.6–2.9 Å for full-length GAC from Toxoplasma gondii in native and selenomethionine-labelled forms. These crystals belong to space group P212121; cell dimensions are roughly a = 119 Å, b = 123 Å, c = 221 Å, α = 90°, β = 90° and γ = 90° with 1 molecule per asymmetric unit, suggesting a more compact conformation than previously proposed
Nagaraj M, Najarzadeh Z, Pansieri J, et al., 2022, Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation, CHEMICAL SCIENCE, Vol: 13, Pages: 536-553, ISSN: 2041-6520
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- Citations: 7
Rattu P, Glencross F, Mader SL, et al., 2022, Atomistic level characterisation of ssDNA translocation through the <i>E</i>. <i>coli</i> proteins CsgG and CsgF for nanopore sequencing (vol 19, pg 6417, 2021), COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL, Vol: 20, Pages: 1027-1027, ISSN: 2001-0370
Kumar A, Zhang X, Vadas O, et al., 2021, Secondary structure and X-Ray crystallographic analysis of the Glideosome-Associated Connector (GAC) from toxoplasma gondii, Publisher: Preprints
A model for parasitic motility has been proposed in which parasite filamentous actin (F-actin) is attached to surface adhesins by a large component of the glideosome, known as the glideosome-associated connector protein (GAC). This large 286 kDa protein interacts at the cytoplasmic face of the plasma membrane with the phosphatidic acid-enriched inner leaflet and cytosolic tails of surface adhesins to connect them to the parasite actomyosin system. GAC is observed initially to the conoid at the apical pole and re-localised with the glideosome to the basal pole in gliding parasite. GAC presumably functions in force transmission to surface adhesins in the plasma membrane and not in force generation. Proper connection between F-actin and the adhesins is as important for motility and invasion as motor operation itself. This notion highlights the need for new structural information on GAC interactions, which has eluded the field since its discovery. We have obtained crystals that diffracted to 2.6-2.9 Å for full-length GAC from Toxoplasma gondii in native and selenomethionine-labelled forms. These crystals belong to space group P212121, cell dimensions are roughly a=119 Å, b=123Å, c=221Å, α=90, β=90, γ=90 with 1 molecule per asymmetric unit, suggesting a more compact conformation than previously proposed.
Liu B, Li S, Liu Y, et al., 2021, Bacteriophage Twort protein Gp168 is a β-clamp inhibitor by occupying the DNA sliding channel, NUCLEIC ACIDS RESEARCH, Vol: 49, Pages: 11367-11378, ISSN: 0305-1048
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- Citations: 4
McKenna S, Giblin SP, Bunn RA, et al., 2021, A highly efficient method for the production and purification of recombinant human CXCL8, PLoS One, Vol: 16, Pages: 1-12, ISSN: 1932-6203
Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.
Kelsall IR, McCrory EH, Xu Y, et al., 2021, HOIL-1-catalysed ubiquitylation of unbranched glucosaccharides and its activation by ubiquitin oligomers
<jats:title>Abstract</jats:title><jats:p>HOIL-1, a component of the Linear Ubiquitin Assembly Complex (LUBAC), ubiquitylates serine and threonine residues in proteins, forming ester bonds (Kelsall et al, 2019, PNAS 116, 13293-13298). Here we report that mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant accumulate polyglucosan in brain, cardiac muscle and other organs, indicating that HOIL-1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL-1 monoubiquitylates glycogen and α1:4-linked maltoheptaose <jats:italic>in vitro</jats:italic> and identify the C6 hydroxyl moiety of glucose as the site of ester-linked ubiquitylation. The HOIL-1-catalysed monoubiquitylation of maltoheptaose was accelerated >100-fold by Met1-linked or Lys63-linked ubiquitin oligomers, which interact with the catalytic RBR domain of HOIL-1. HOIL-1 also transferred preformed ubiquitin oligomers to maltoheptaose <jats:italic>en bloc</jats:italic>, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL-1, bound to amylose resin <jats:italic>in vitro</jats:italic>, suggesting a potential function in targeting HOIL-1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal by glycophagy to prevent precipitation as polyglucosan.</jats:p>
Wang Z, Wang H, Mulvenna N, et al., 2021, A bacteriophage DNA mimic protein employs a non-specific strategy to inhibit the bacterial RNA polymerase, Frontiers in Microbiology, Vol: 12, Pages: 1-10, ISSN: 1664-302X
DNA mimicry by proteins is a strategy that employed by some proteins to occupy the binding sites of the DNA-binding proteins and deny further access to these sites by DNA. Such proteins have been found in bacteriophage, eukaryotic virus, prokaryotic, and eukaryotic cells to imitate non-coding functions of DNA. Here, we report another phage protein Gp44 from bacteriophage SPO1 of Bacillus subtilis, employing mimicry as part of unusual strategy to inhibit host RNA polymerase. Consisting of three simple domains, Gp44 contains a DNA binding motif, a flexible DNA mimic domain and a random-coiled domain. Gp44 is able to anchor to host genome and interact bacterial RNA polymerase via the β and β′ subunit, resulting in bacterial growth inhibition. Our findings represent a non-specific strategy that SPO1 phage uses to target different bacterial transcription machinery regardless of the structural variations of RNA polymerases. This feature may have potential applications like generation of genetic engineered phages with Gp44 gene incorporated used in phage therapy to target a range of bacterial hosts.
Wang Z, Zhao S, Li Y, et al., 2021, RssB-mediated σ<SUP>S</SUP> Activation is Regulated by a Two-Tier Mechanism via Phosphorylation and Adaptor Protein - IraD, JOURNAL OF MOLECULAR BIOLOGY, Vol: 433, ISSN: 0022-2836
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- Citations: 3
Rattu P, Glencross F, Mader SL, et al., 2021, Atomistic level characterisation of ssDNA translocation through the <i>E. coli</i> proteins CsgG and CsgF for nanopore sequencing, COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL, Vol: 19, Pages: 6417-6430, ISSN: 2001-0370
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- Citations: 2
Darby JF, Vidler LR, Simpson PJ, et al., 2020, Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches, SCIENTIFIC REPORTS, Vol: 10, ISSN: 2045-2322
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- Citations: 5
Murphy P, Xu Y, Rouse SL, et al., 2020, Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer, NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723
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- Citations: 7
Broncel M, Dominicus C, Vigetti L, et al., 2020, Profiling of myristoylation in <i>Toxoplasma</i> <i>gondii</i> reveals an <i>N</i>-myristoylated protein important for host cell penetration, ELIFE, Vol: 9, ISSN: 2050-084X
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- Citations: 12
Sewell L, Stylianou F, Xu Y, et al., 2020, NMR insights into the pre-amyloid ensemble and secretion targeting of the curli subunit CsgA, Scientific Reports, Vol: 10, ISSN: 2045-2322
The biofilms of Enterobacteriaceae are fortified by assembly of curli amyloid fibres on the cell surface. Curli not only provides structural reinforcement, but also facilitates surface adhesion. To prevent toxic intracellular accumulation of amyloid precipitate, secretion of the major curli subunit, CsgA, is tightly regulated. In this work, we have employed solution state NMR spectroscopy to characterise the structural ensemble of the pre-fibrillar state of CsgA within the bacterial periplasm, and upon recruitment to the curli pore, CsgG, and the secretion chaperone, CsgE. We show that the N-terminal targeting sequence (N) of CsgA binds specifically to CsgG and that its subsequent sequestration induces a marked transition in the conformational ensemble, which is coupled to a preference for CsgE binding. These observations lead us to suggest a sequential model for binding and structural rearrangement of CsgA at the periplasmic face of the secretion machinery.
Sheppard D, Berry J-L, Denise R, et al., 2020, The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold, Journal of Biological Chemistry, Vol: 295, Pages: 6594-6604, ISSN: 0021-9258
<jats:p>Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, <jats:italic>Streptococcus sanguinis</jats:italic> (ComGC<jats:sub>SS</jats:sub>) and <jats:italic>Streptococcus pneumoniae</jats:italic> (ComGC<jats:sub>SP</jats:sub>), revealing that this pilin displays extensive structural conservation. Strikingly, ComGC<jats:sub>SS</jats:sub> and ComGC<jats:sub>SP</jats:sub> exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.</jats:p>
Chorev DS, Tang H, Rouse SL, et al., 2020, The use of sonicated lipid vesicles for mass spectrometry of membrane protein complexes, NATURE PROTOCOLS, Vol: 15, Pages: 1690-1706, ISSN: 1754-2189
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- Citations: 25
Matthews S, McKenna S, Malito E, et al., 2020, Structure, dynamics and immunogenicity of a catalytically inactive CXC Chemokine-degrading Protease SpyCEP from Streptococcus pyogenes, Computational and Structural Biotechnology Journal, Vol: 18, Pages: 650-660, ISSN: 2001-0370
Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.
Murphy P, Xu Y, Rouse SL, et al., 2019, Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer
<jats:title>Abstract</jats:title><jats:p>Post-translational modification of proteins with ubiquitin represents a widely used mechanism for cellular regulation. Ubiquitin is activated by an E1 enzyme, transferred to an E2 conjugating enzyme and covalently linked to substrates by one of an estimated 600 E3 ligases (1). RING E3 ligases play a pivotal role in selecting substrates and priming the ubiquitin loaded E2 (E2~Ub) for catalysis (2,3). RING E3 RNF4 is a SUMO targeted ubiquitin ligase (4) with important roles in arsenic therapy for cancer (4,5) and in DNA damage responses (6,7). RNF4 has a RING domain and a substrate recognition domain containing multiple SUMO Interaction Motifs (SIM<jats:sc>s</jats:sc>) embedded in a region thought to be intrinsically disordered (8). While molecular details of SUMO recognition by the SIMs (8–10) and RING engagement of ubiquitin loaded E2 (3,11–15) have been determined, the mechanism by which SUMO substrate is delivered to the RING to facilitate ubiquitin transfer is an important question to be answered. Here, we show that the intrinsically disordered substrate-recognition domain of RNF4 maintains the SIMs in a compact global architecture that facilitates SUMO binding, while a highly-basic region positions substrate for nucleophilic attack on RING-bound ubiquitin loaded E2. Contrary to our expectation that the substrate recognition domain of RNF4 was completely disordered, distance measurements using single molecule Fluorescence Resonance Energy Transfer (smFRET) and NMR paramagnetic relaxation enhancement (PRE) revealed that it adopts a defined conformation primed for SUMO interaction. Mutational and biochemical analysis indicated that electrostatic interactions involving the highly basic region linking the substrate recognition and RING domains juxtaposed those regions and mediated substrate ubiquitination. Our results offer insight into a key step in substrate ubiquitination by a membe
Wang Z, Zhao S, Jiang S, et al., 2019, Resonance assignments of N-terminal receiver domain of sigma factor S regulator RssB from <i>Escherichia coli</i>, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 13, Pages: 333-337, ISSN: 1874-2718
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- Citations: 1
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