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1.
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
2.
Methods Mol Biol ; 2168: 273-297, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33582997

RESUMO

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10-9-102 s).


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Fluorescência , Proteínas de Membrana/análise , Imagem Individual de Molécula/métodos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Conformação Proteica
3.
Methods Enzymol ; 607: 93-130, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30149870

RESUMO

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.


Assuntos
Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Pirofosfatases/metabolismo , Imagem Individual de Molécula/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Desenho de Fármacos , Ensaios Enzimáticos/instrumentação , Transferência Ressonante de Energia de Fluorescência/instrumentação , Corantes Fluorescentes/química , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Mutagênese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Pirofosfatases/química , Pirofosfatases/genética , Pirofosfatases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Alinhamento de Sequência , Imagem Individual de Molécula/instrumentação , Software
4.
J Am Chem Soc ; 139(21): 7156-7159, 2017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28497688

RESUMO

Synthetic molecular motors continue to attract great interest due to their ability to transduce energy into nanomechanical motion, the potential to do work and drive systems out-of-equilibrium. Of particular interest are unidirectional rotary molecular motors driven by chemical fuel or light. Probing the mechanistic details of their operation at the single-molecule level is hampered by the diffraction limit, which prevents the collection of dynamic positional information by traditional optical methods. Here, we use defocused wide-field imaging to examine the unidirectional rotation of individual molecular rotary motors on a quartz surface in unprecedented detail. The sequential occupation of nanomechanical states during the UV and heat-induced cycle of rotation are directly imaged in real-time. The approach will undoubtedly prove important in elucidating the mechanistic details and assessing the utility of novel synthetic molecular motors in the future.

5.
Chem Soc Rev ; 45(22): 6118-6129, 2016 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-26932423

RESUMO

Nature's molecular machines are a constant source of inspiration to the chemist. Many of these molecular machines function within lipid membranes, allowing them to exploit potential gradients between spatially close, but chemically distinct environments to fuel their work cycle. Indeed, the realisation of such principles in synthetic transmembrane systems remains a tantalising goal. This tutorial review opens by highlighting seminal examples of synthetic molecular machines. We illustrate the importance of surfaces for facilitating the extraction of work from molecular switches and motors. We chart the development of man-made transmembrane systems; from passive to machine-like stimuli-responsive channels, to fully autonomous transmembrane molecular machines. Finally, we highlight higher-order compartmentalised systems that exhibit emergent properties. We suggest that such higher-order architectures could serve as platforms for sophisticated devices that co-ordinate the activity of numerous transmembrane molecular machines.


Assuntos
Nanoestruturas/química , Nanotecnologia , Membranas Artificiais , Estrutura Molecular
6.
Angew Chem Int Ed Engl ; 55(4): 1345-9, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26661295

RESUMO

Biological molecular machines operate far from equilibrium by coupling chemical potential to repeated cycles of dissipative nanomechanical motion. This principle has been exploited in supramolecular systems that exhibit true machine behavior in solution and on surfaces. However, designed membrane-spanning assemblies developed to date have been limited to simple switches or stochastic shuttles, and true machine behavior has remained elusive. Herein, we present a transmembrane nanoactuator that turns over chemical fuel to drive autonomous reciprocating (back-and-forth) nanomechanical motion. Ratcheted reciprocating motion of a DNA/PEG copolymer threaded through a single α-hemolysin pore was induced by a combination of DNA strand displacement processes and enzyme-catalyzed reactions. Ion-current recordings revealed saw-tooth patterns, indicating that the assemblies operated in autonomous, asymmetric cycles of conformational change at rates of up to one cycle per minute.


Assuntos
Membranas Artificiais , Nanotecnologia , DNA/química , Polietilenoglicóis/química
7.
Chem Commun (Camb) ; 51(61): 12243-6, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26135014

RESUMO

Here we investigate the modulation of solvent isotope effects by the entry of DNA molecules into individual α-haemolysin nanopores. Solvent isotope effects in D2O versus H2O were enhanced (kH/kD ≈ 1.6) compared to the bulk (kH/kD ≈ 1.2), except when the pore was most blocked (kH/kD ≤ 1.1).


Assuntos
DNA de Cadeia Simples/química , Óxido de Deutério/química , Proteínas Hemolisinas/química , Nanoporos , Solventes/química , Água/química
8.
Langmuir ; 26(20): 15920-4, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20853821

RESUMO

A straightforward functionalization of a titanium surface using "click" chemistry is reported. A "clickable" titanium surface platform was prepared by the immobilization of an azide-functionalized electroactive catechol anchor and was subsequently derivatized with an electroactive or fluorinated probe via the CuAAC (copper-catalyzed azide-alkyne cycloaddition) reaction. The course of the reaction was investigated by contact angle, XPS, and electrochemical measurements.


Assuntos
Química Click , Titânio/química , Alcinos/química , Azidas/química , Catálise , Catecóis/química , Cobre/química , Eletroquímica , Espectroscopia Fotoeletrônica , Propriedades de Superfície
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