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1.
Nat Commun ; 12(1): 6721, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34795302

RESUMO

Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE. Here we demonstrate that, in addition, MukB interacts specifically with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction at the joint of the MukB coiled-coil and show that the interaction is necessary for MukB ATPase and for MukBEF function in vivo.

3.
PLoS One ; 16(10): e0256070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34653190

RESUMO

Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 µM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 µM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/metabolismo , Haemophilus influenzae/metabolismo , Heme/metabolismo , RNA de Cadeia Dupla/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Proteínas de Transporte/genética , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Otite Média/microbiologia , Otite Média/patologia , Conformação Proteica , Transporte Proteico/fisiologia , RNA de Cadeia Dupla/genética , Motivos de Ligação ao RNA/genética , Fatores de Virulência/metabolismo
4.
Structure ; 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34492227

RESUMO

R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.

5.
Chem Commun (Camb) ; 57(82): 10747-10750, 2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34585198

RESUMO

We developed a native mass spectrometry-based approach to quantify the monomer-dimer equilibrium of the LPS transport protein LptH. We use this method to assess the potency and efficacy of an antimicrobial peptide and small molecule disruptors, obtaining new information on their structure-activity relationships. This approach led to the identification of quinoline-based hit compounds representing the basis for the development of novel LPS transport inhibitors.

6.
Elife ; 102021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34585666

RESUMO

Structural Maintenance of Chromosomes (SMC) complexes have ubiquitous roles in compacting DNA linearly, thereby promoting chromosome organization-segregation. Interaction between the Escherichia coli SMC complex, MukBEF, and matS-bound MatP in the chromosome replication termination region, ter, results in depletion of MukBEF from ter, a process essential for efficient daughter chromosome individualization and for preferential association of MukBEF with the replication origin region. Chromosome-associated MukBEF complexes also interact with topoisomerase IV (ParC2E2), so that their chromosome distribution mirrors that of MukBEF. We demonstrate that MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding, which occurs with the same dimer to dimer stoichiometry. Furthermore, we show that matS DNA competes with the MukB hinge for MatP binding. Cells expressing MukBEF complexes that are mutated at the ParC/MatP binding interface are impaired in ParC binding and have a mild defect in MukBEF function. These data highlight competitive binding as a means of globally regulating MukBEF-topoisomerase IV activity in space and time.


Assuntos
Ligação Competitiva , Proteínas Cromossômicas não Histona/química , DNA Topoisomerase IV/química , Proteínas de Escherichia coli/química , Escherichia coli/química
7.
mBio ; 12(5): e0178721, 2021 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-34544275

RESUMO

Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter. IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.

8.
J Mol Biol ; 433(21): 167226, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34487790

RESUMO

Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure-function studies of metalloid porins, including the basis of their substrate selectivity.


Assuntos
Aquaporinas/química , Proteínas de Arabidopsis/química , Oryza/metabolismo , Raízes de Plantas/metabolismo , Ácido Silícico/metabolismo , Silício/metabolismo , Sequência de Aminoácidos , Aquaporinas/genética , Aquaporinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Difusão , Expressão Gênica , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Oryza/genética , Raízes de Plantas/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ácido Silícico/química , Silício/química , Especificidade por Substrato
9.
JACS Au ; 1(8): 1147-1157, 2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34462738

RESUMO

The SARS-CoV-2 nucleocapsid (N) protein is a highly immunogenic viral protein that plays essential roles in replication and virion assembly. Here, using native mass spectrometry, we show that dimers are the functional unit of ribonucleoprotein assembly and that N protein binds RNA with a preference for GGG motifs, a common motif in coronavirus packaging signals. Unexpectedly, proteolytic processing of N protein resulted in the formation of additional proteoforms. The N-terminal proteoforms bind RNA, with the same preference for GGG motifs, and bind to cyclophilin A, an interaction which can be abolished by approved immunosuppressant cyclosporin A. Furthermore, N proteoforms showed significantly different interactions with IgM, IgG, and IgA antibodies from convalescent plasma. Notably, the C-terminal proteoform exhibited a heightened interaction with convalescent antibodies, suggesting the antigenic epitope is localized to the C-terminus. Overall, the different interactions of N proteoforms highlight potential avenues for therapeutic intervention and identify a stable and immunogenic proteoform as a possible candidate for immune-directed therapies.

10.
Sci Adv ; 7(34)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34417180

RESUMO

Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

11.
Anal Chem ; 93(30): 10435-10443, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34279906

RESUMO

Glycoproteins are inherently heterogeneous and therefore resolving structures in their entirety remains a major challenge in structural biology. Native mass spectrometry has transformed our ability to study glycoproteins, and despite advances in high-resolution instrumentation, there are comparatively a few studies demonstrating its potential with data largely limited to an overall measure of monosaccharide composition for all glycans across glycosylation sites for a given protein. Clearly, these readouts lack glycan topology information, namely, monosaccharide linkage and glycan branching. To address this deficiency, we developed a new approach that joins native mass spectrometry with glycan exoglycosidase sequencing, the combination of which provides remarkable glycoprotein structural details. We show how N-glycan branching, terminal fucosylation, LacNAc extensions, and N- and O-glycan occupancy (i.e., total number of glycans) can be directly characterized on intact glycoproteins with minimal sample preparation. Taken together, native exoglycosidase sequencing mass spectrometry (NES-MS) notably improves our ability to characterize protein glycosylation, addressing a significant need in structural biology that will enable new routes to understand glycoprotein function.


Assuntos
Glicômica , Glicoproteínas , Glicoproteínas/metabolismo , Glicosilação , Espectrometria de Massas , Polissacarídeos
12.
Nat Commun ; 12(1): 4349, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272394

RESUMO

Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.


Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismo
13.
Nat Commun ; 12(1): 4625, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330923

RESUMO

Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.


Assuntos
Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Canais Iônicos/metabolismo , Klebsiella/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Transporte Biológico , Microscopia Crioeletrônica/métodos , Canais Iônicos/química , Canais Iônicos/ultraestrutura , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica
14.
Proc Natl Acad Sci U S A ; 118(31)2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34321357

RESUMO

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam-milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.

15.
Bioinformatics ; 2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145888

RESUMO

MOTIVATION: Native mass spectrometry is now a well-established method for the investigation of protein complexes, specifically their subunit stoichiometry and ligand binding properties. Recent advances allowing the analysis of complex mixtures lead to an increasing diversity and complexity in the spectra obtained. These spectra can be time consuming to tackle through manual assignment and challenging for automated approaches. RESULTS: Native Mass Spectrometry Visual Analyser (NaViA) is a web-based tool to augment the manual process of peak assignment. In addition to matching masses to the stoichiometry of its component subunits it allows raw data processing, assignment and annotation and permits mass spectra to be shared with their respective interpretation. AVAILABILITY: NaViA is open-source and can be accessed online under https://navia.ms. The source code and documentation can be accessed at https://github.com/d-que/navia, under the BSD 2-Clause license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

16.
Curr Opin Struct Biol ; 70: 53-60, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33964676

RESUMO

Membrane proteins are important macromolecules that play crucial roles in many cellular and physiological processes. Over the past two decades, the use of mass spectrometry as a biophysical tool to characterise membrane proteins has grown steadily. By capturing these dynamic complexes in the gas phase, many unknown small molecule interactions have been revealed. One particular application of this research has been the focus on antibiotic resistance with considerable efforts being made to understand underlying mechanisms. Here we review recent advances in the application of mass spectrometry that have yielded both structural and dynamic information on the interactions of antibiotics with proteins involved in bacterial cell envelope biogenesis and drug efflux.


Assuntos
Proteínas de Membrana , Preparações Farmacêuticas , Transporte Biológico , Lipídeos , Espectrometria de Massas , Proteínas de Membrana/metabolismo
17.
EMBO J ; 40(14): e107294, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031912

RESUMO

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.


Assuntos
Cloretos/metabolismo , Nucleotídeos/metabolismo , Potássio/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular , Tamanho Celular , Humanos , Fosforilação/fisiologia , Células Sf9 , Transdução de Sinais/fisiologia
18.
Nature ; 593(7857): 125-129, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33854236

RESUMO

Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis1-3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets4,5. The natural compound darobactin targets the bacterial insertase BamA6-the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins7-13. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid ß-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenho de Fármacos , Escherichia coli/citologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
19.
Curr Opin Struct Biol ; 69: 78-85, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33930613

RESUMO

Structure determination of membrane proteins has highlighted the many roles played by lipids in influencing overall protein architecture. It is now widely accepted that lipids surrounding membrane proteins play crucial roles by modulating their conformational, structural, and functional properties. Capturing often transient lipid interactions and defining their chemical identity, however, remains challenging. Recent advances in mass spectrometry have resolved questions concerning lipid interactions by providing the molecular composition of intact complexes in association with lipids. Together with other biophysical tools, a picture is emerging of the dynamic nature of lipid-mediated interactions and their effects on conformation, interactions, and signaling.


Assuntos
Lipídeos de Membrana , Proteínas de Membrana , Membrana Celular , Espectrometria de Massas
20.
Nano Lett ; 21(7): 2824-2831, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33787280

RESUMO

Integral membrane proteins pose considerable challenges to mass spectrometry (MS) owing to the complexity and diversity of the components in their native environment. Here, we use native MS to study the post-translational maturation of bacteriorhodopsin (bR) and archaerhodopsin-3 (AR3), using both octyl-glucoside detergent micelles and lipid-based nanoparticles. A lower collision energy was required to obtain well-resolved spectra for proteins in styrene-maleic acid copolymer (SMA) Lipodisqs than in membrane scaffold protein (MSP) Nanodiscs. By comparing spectra of membrane proteins prepared using the different membrane mimetics, we found that SMA may favor selective solubilization of correctly folded proteins and better preserve native lipid interactions than other membrane mimetics. Our spectra reveal the correlation between the post-translation modifications (PTMs), lipid-interactions, and protein-folding states of bR, providing insights into the process of maturation of the photoreceptor proteins.


Assuntos
Proteínas de Membrana , Nanopartículas , Bicamadas Lipídicas , Lipídeos , Espectrometria de Massas , Micelas
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