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1.
Proc Natl Acad Sci U S A ; 118(41)2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34615715

RESUMO

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

2.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
3.
Commun Biol ; 3(1): 766, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318620

RESUMO

The ß-barrel assembly machinery (BAM) catalyses the folding and insertion of ß-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The ß-barrel domain of BamA is in a 'lateral open' conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM's lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid 'disruptase' activity of BAM, suggested to be an important part of its functional mechanism.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas , Lipídeos , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Nanoestruturas , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/metabolismo , Catálise , Complexos Multiproteicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteolipídeos/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(41): 25523-25531, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32999060

RESUMO

Antibiotic resistance in clinically important bacteria can be mediated by target protection mechanisms, whereby a protein binds to the drug target and protects it from the inhibitory effects of the antibiotic. The most prevalent source of clinical resistance to the antibiotic fusidic acid (FA) is expression of the FusB family of proteins that bind to the drug target (Elongation factor G [EF-G]) and promote dissociation of EF-G from FA-stalled ribosome complexes. FusB binding causes changes in both the structure and conformational flexibility of EF-G, but which of these changes drives FA resistance was not understood. We present here detailed characterization of changes in the conformational flexibility of EF-G in response to FusB binding and show that these changes are responsible for conferring FA resistance. Binding of FusB to EF-G causes a significant change in the dynamics of domain III of EF-GC3 that leads to an increase in a minor, more disordered state of EF-G domain III. This is sufficient to overcome the steric block of transmission of conformational changes within EF-G by which FA prevents release of EF-G from the ribosome. This study has identified an antibiotic resistance mechanism mediated by allosteric effects on the dynamics of the drug target.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias , Farmacorresistência Bacteriana/fisiologia , Ácido Fusídico/farmacologia , Fator G para Elongação de Peptídeos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , Domínios Proteicos
5.
Protein Sci ; 29(8): 1851-1857, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32557917

RESUMO

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yield inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly populated "excited" states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein, and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualization of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication.


Assuntos
Modelos Moleculares , Proteínas/química , Software , Conformação Proteica
6.
Nat Chem Biol ; 16(9): 1019-1025, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32572278

RESUMO

The ß-barrel assembly machinery (BAM) inserts outer membrane ß-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF-OMP complexes. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. RcsF is lodged deep within the lumen of the BamA barrel, binding regions proposed to undergo outward and lateral opening during OMP insertion. On the basis of our structural and biochemical data, we propose a push-and-pull model for RcsF export following conformational cycling of BamA, and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Conformação Proteica
7.
Nat Commun ; 11(1): 2155, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358557

RESUMO

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Proteínas de Transporte/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Espectrometria de Massas , Chaperonas Moleculares/genética , Peptidilprolil Isomerase/genética , Ligação Proteica
8.
Methods Mol Biol ; 2168: 233-261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33582995

RESUMO

Native mass spectrometry and native ion mobility mass spectrometry are now established techniques in structural biology, with recent work developing these methods for the study of integral membrane proteins reconstituted in both lipid bilayer and detergent environments. Here we show how native mass spectrometry can be used to interrogate integral membrane proteins, providing insights into conformation, oligomerization, subunit composition/stoichiometry, and interactions with detergents/lipids/drugs. Furthermore, we discuss the sample requirements and experimental considerations unique to integral membrane protein native mass spectrometry research.


Assuntos
Membrana Celular/metabolismo , Detergentes/química , Espectrometria de Mobilidade Iônica/métodos , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Humanos , Conformação Proteica
9.
Elife ; 82019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31552823

RESUMO

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human ß2-microglobulin (ß2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-ß structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.


Assuntos
Amiloide/química , Amiloide/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Multimerização Proteica , Microglobulina beta-2/química , Microglobulina beta-2/metabolismo , Fenômenos Biofísicos , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Ligação Proteica
10.
Mol Cell ; 74(4): 729-741.e7, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30982745

RESUMO

The nascent polypeptide-associated complex (NAC) is a conserved ribosome-associated protein biogenesis factor. Whether NAC exerts chaperone activity and whether this function is restricted to de novo protein synthesis is unknown. Here, we demonstrate that NAC directly exerts chaperone activity toward structurally diverse model substrates including polyglutamine (PolyQ) proteins, firefly luciferase, and Aß40. Strikingly, we identified the positively charged ribosome-binding domain in the N terminus of the ßNAC subunit (N-ßNAC) as a major chaperone entity of NAC. N-ßNAC by itself suppressed aggregation of PolyQ-expanded proteins in vitro, and the positive charge of this domain was critical for this activity. Moreover, we found that NAC also exerts a ribosome-independent chaperone function in vivo. Consistently, we found that a substantial fraction of NAC is non-ribosomal bound in higher eukaryotes. In sum, NAC is a potent suppressor of aggregation and proteotoxicity of mutant PolyQ-expanded proteins associated with human diseases like Huntington's disease and spinocerebellar ataxias.


Assuntos
Peptídeos beta-Amiloides/genética , Chaperonas Moleculares/genética , Agregação Patológica de Proteínas/genética , Peptídeos beta-Amiloides/química , Sítios de Ligação/genética , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Luciferases/química , Luciferases/genética , Chaperonas Moleculares/química , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Biossíntese de Proteínas/genética , Domínios Proteicos/genética , Dobramento de Proteína , Ribossomos/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia
11.
J Mol Biol ; 431(6): 1267-1283, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30716334

RESUMO

SurA is a conserved ATP-independent periplasmic chaperone involved in the biogenesis of outer-membrane proteins (OMPs). Escherichia coli SurA has a core domain and two peptidylprolyl isomerase (PPIase) domains, the role(s) of which remain unresolved. Here we show that while SurA homologues in early proteobacteria typically contain one or no PPIase domains, the presence of two PPIase domains is common in SurA in later proteobacteria, implying an evolutionary advantage for this domain architecture. Bioinformatics analysis of >350,000 OMP sequences showed that their length, hydrophobicity and aggregation propensity are similar across the proteobacterial classes, ruling out a simple correlation between SurA domain architecture and these properties of OMP sequences. To investigate the role of the PPIase domains in SurA activity, we deleted one or both PPIase domains from E.coli SurA and investigated the ability of the resulting proteins to bind and prevent the aggregation of tOmpA (19 kDa) and OmpT (33 kDa). The results show that wild-type SurA inhibits the aggregation of both OMPs, as do the cytoplasmic OMP chaperones trigger factor and SecB. However, while the ability of SurA to bind and prevent tOmpA aggregation does not depend on its PPIase domains, deletion of even a single PPIase domain ablates the ability of SurA to prevent OmpT aggregation. The results demonstrate that the core domain of SurA endows its generic chaperone ability, while the presence of PPIase domains enhances its chaperone activity for specific OMPs, suggesting one reason for the conservation of multiple PPIase domains in SurA in proteobacteria.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Peptídeo Hidrolases/metabolismo , Peptidilprolil Isomerase/química , Domínios Proteicos , Proteínas da Membrana Bacteriana Externa/química , Fenômenos Biofísicos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Proteobactérias/metabolismo , Deleção de Sequência
12.
Angew Chem Int Ed Engl ; 57(51): 16688-16692, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30393918

RESUMO

Analysing protein complexes by chemical crosslinking-mass spectrometry (XL-MS) is limited by the side-chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue-unbiased diazirine-based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol-containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this "tag and transfer" approach is demonstrated using a well-defined peptide/protein regulatory interaction (BID80-102 /MCL-1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).


Assuntos
Reagentes para Ligações Cruzadas/química , Cisteína/química , Mesilatos/química , Mapas de Interação de Proteínas , Proteínas/química , Espectrometria de Massas , Estrutura Molecular , Processos Fotoquímicos
13.
Nucleic Acids Res ; 46(15): 7924-7937, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-29796667

RESUMO

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.


Assuntos
Chaperonas Moleculares/metabolismo , Orthoreovirus Aviário/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Bases , Genoma Viral/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Orthoreovirus Aviário/genética , Ligação Proteica , Estrutura Secundária de Proteína , RNA Viral/química , RNA Viral/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
14.
Methods ; 147: 187-205, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29510247

RESUMO

The last ∼25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.


Assuntos
Espectrometria de Massas/métodos , Proteínas de Membrana/química , Detergentes/química , Conformação Proteica , Dobramento de Proteína
15.
J Mol Biol ; 429(23): 3776-3792, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-28919234

RESUMO

The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the ß-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA ß-barrel domain, but is greater when full-length BamA is present, indicating that both the ß-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Membrana Celular/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Periplasma/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/metabolismo , Biocatálise , Membrana Celular/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Lipossomos/metabolismo , Simulação de Dinâmica Molecular
16.
Anal Chem ; 89(17): 8844-8852, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28726379

RESUMO

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion-substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein's conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.


Assuntos
Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Espectrometria de Massas , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cisteína/química , Etilmaleimida/química , Hidantoínas/química , Hidantoínas/metabolismo , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Micrococcaceae/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Sódio/química , Sódio/metabolismo , Especificidade por Substrato
17.
J Am Soc Mass Spectrom ; 28(9): 1855-1862, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28484973

RESUMO

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein's size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstract ᅟ.


Assuntos
Espectrometria de Mobilidade Iônica/métodos , Proteínas/química , RNA/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Gases/química
18.
J Am Soc Mass Spectrom ; 28(1): 50-55, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27343183

RESUMO

Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-ß-maltoside (DDM) detergent micelles. Using quantitative mass spectrometry analyses, we show that fast photochemical oxidation reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of additional intermolecular contacts. Graphical Abstract ᅟ.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Detergentes/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Glucosídeos/química , Peptídeo Hidrolases/química , Polímeros/química , Propilaminas/química , Cromatografia Líquida/métodos , Peróxido de Hidrogênio/química , Radical Hidroxila/química , Micelas , Modelos Moleculares , Oxirredução , Processos Fotoquímicos , Fotólise , Solubilidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
19.
Nat Commun ; 7: 12865, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27686148

RESUMO

The ß-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA-E), which is essential for viability in E. coli. BAM promotes the folding and insertion of ß-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a 'lateral gating' motion of the ß-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.

20.
Nat Struct Mol Biol ; 23(9): 786-793, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27455461

RESUMO

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry-mass spectrometry (IMS-MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Cinética , Chaperonas Moleculares/fisiologia , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína
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