RESUMO
Proximity-enhanced chemical cross-linking is an invaluable tool for probing protein-protein interactions and enhancing the potency of potential peptide and protein drugs. Here, we extend this approach to covalently stabilize large macromolecular assemblies. We used SuFEx chemistry to covalently stabilize an 18-subunit pore-forming complex, CsgG:CsgF, consisting of nine CsgG membrane protein subunits that noncovalently associate with nine CsgF peptides. Derivatives of the CsgG:CsgF pore have been used for DNA sequencing, which places high demands on the structural stability and homogeneity of the complex. To increase the robustness of the pore, we designed and synthesized derivatives of CsgF-bearing sulfonyl fluorides, which react with CsgG in very high yield to form a covalently stabilized CsgG:CsgF complex. The resulting pores formed highly homogeneous channels when added to artificial membranes. The high yield and rapid reaction rate of the SuFEx reaction prompted molecular dynamics simulations, which revealed that the SO2F groups in the initially formed complex are poised for nucleophilic reaction with a targeted Tyr. These results demonstrate the utility of SuFEx chemistry to structurally stabilize very large (here, 280 kDa) assemblies.
RESUMO
Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanoporos , RNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Análise de Sequência de RNA/métodosRESUMO
Single-molecule long-read DNA sequencing with biological nanopores is fast and high-throughput but suffers reduced accuracy in homonucleotide stretches. We now combine the CsgG nanopore with the 35-residue N-terminal region of its extracellular interaction partner CsgF to produce a dual-constriction pore with improved signal and base-calling accuracy for homopolymer regions. The electron cryo-microscopy structure of CsgG in complex with full-length CsgF shows that the 33 N-terminal residues of CsgF bind inside the ß-barrel of the pore, forming a defined second constriction. In complexes of CsgG bound to a 35-residue CsgF constriction peptide, the second constriction is separated from the primary constriction by ~25 Å. We find that both constrictions contribute to electrical signal modulation during single-stranded DNA translocation. DNA sequencing using a prototype CsgG-CsgF protein pore with two constrictions improved single-read accuracy by 25 to 70% in homopolymers up to 9 nucleotides long.
Assuntos
Nanoporos , Nucleotídeos/genética , Sequência de Bases , Microscopia Crioeletrônica , DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Modelos MolecularesRESUMO
The dispersion of carbon nanotubes (CNTs) in aqueous media is of potential importance in a number of biomedical applications. CNT solubilization has been achieved via the non-covalent adsorption of lipids and detergent onto the tube surface. We use coarse-grained molecular dynamics to study the self-assembly of CNTs with various amphiphiles, namely a bilayer-forming lipid, dipalmitoylphosphatidylcholine (DPPC), and two species of detergent, dihexanoylphosphatidylcholine (DHPC) and lysophosphatidylcholine (LPC). We find that for a low amphiphile/CNT ratio, DPPC, DHPC and LPC all wrap around the CNT. Upon increasing the number of amphiphiles, a transition in adsorption is observed: DPPC encapsulates the CNT within a cylindrical micelle, whilst both DHPC and LPC adsorb onto CNTs in hemimicelles. This study highlights differences in adsorption mechanism of bilayer-forming lipids and detergents on CNTs which may in the future be exploitable to enable enhancement of CNT solubilization whilst minimizing perturbation of cell membrane integrity.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Detergentes/química , Modelos Químicos , Nanotubos de Carbono/química , Adsorção , Simulação por Computador , Lisofosfatidilcolinas/química , Fosfatidilcolinas/químicaRESUMO
Coarse-grained molecular dynamics simulations have been used to explore the interactions of C(60) and its derivatives with lipid bilayers. Pristine C(60) partitions into the bilayer core, whilst C(60)(OH)(20) experiences a central energetic barrier to permeation across the bilayer. For intermediate levels of derivatization, e.g. C(60)(OH)(10), this central barrier is smaller and there is an energetic well at the bilayer/water interface, thus promoting entry into cells via bilayer permeation whilst maintaining solubility in water.
Assuntos
Simulação por Computador , Fulerenos/química , Bicamadas Lipídicas/química , Modelos Moleculares , TermodinâmicaRESUMO
The accurate sequencing of DNA using nanopores requires control over the speed of DNA translocation through the pores and also of the DNA conformation. Our studies show that ssDNA translocates through hourglass-shaped pores with hydrophobic constriction regions when an electric field is applied. The constriction provides a barrier to translocation and thereby slows down DNA movement through the pore compared with pores without the constriction. We show that ssDNA moves through these hydrophobic pores in an extended conformation and therefore does not form undesirable secondary structures that may affect the accuracy of partial current blockages for DNA sequencing.
Assuntos
DNA de Cadeia Simples/química , Nanoporos , Sequência de Bases , Eletricidade , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Conformação de Ácido NucleicoRESUMO
It is desirable that nanopores that are components of biosensors are gated, i.e., capable of controllable switching between closed (impermeable) and open (permeable) states. A central hydrophobic barrier within a nanopore may act as a voltage-dependent gate via electrowetting, i.e., changes in nanopore surface wettability by application of an electric field. We use "computational electrophysiology" simulations to demonstrate and characterize electrowetting of a biomimetic nanopore containing a hydrophobic gate. We show that a hydrophobic gate in a model ß-barrel nanopore can be functionally opened by electrowetting at voltages that do not electroporate lipid bilayers. During the process of electrowetting, voltage-induced alignment of water dipoles occurs within the hydrophobic gate region of the nanopore, with water entry preceding permeation of ions through the opened nanopore. When the ionic imbalance that generates a transbilayer potential is dissipated, water is expelled from the hydrophobic gate and the nanopore recloses. The open nanopore formed by electrowetting of a "featureless" ß-barrel is anionic selective due to the transmembrane dipole potential resulting from binding of Na+ ions to the headgroup regions of the surrounding lipid bilayer. Thus, hydrophobic barriers can provide voltage-dependent gates in designed biomimetic nanopores. This extends our understanding of hydrophobic gating in synthetic and biological nanopores, providing a framework for the design of functional nanopores with tailored gating functionality.
Assuntos
Materiais Biomiméticos/química , Eletroumectação , Interações Hidrofóbicas e Hidrofílicas , Nanoporos , Ativação do Canal Iônico , Bicamadas Lipídicas/química , Modelos MolecularesRESUMO
Ion channels play key roles in cell membranes, and recent advances are yielding an increasing number of structures. However, their functional relevance is often unclear and better tools are required for their functional annotation. In sub-nanometer pores such as ion channels, hydrophobic gating has been shown to promote dewetting to produce a functionally closed (i.e., non-conductive) state. Using the serotonin receptor (5-HT3R) structure as an example, we demonstrate the use of molecular dynamics to aid the functional annotation of channel structures via simulation of the behavior of water within the pore. Three increasingly complex simulation analyses are described: water equilibrium densities; single-ion free-energy profiles; and computational electrophysiology. All three approaches correctly predict the 5-HT3R crystal structure to represent a functionally closed (i.e., non-conductive) state. We also illustrate the application of water equilibrium density simulations to annotate different conformational states of a glycine receptor.
Assuntos
Canais Iônicos/química , Receptores 5-HT3 de Serotonina/química , Água/química , Membrana Celular/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a ß-barrel pore â¼10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.
Assuntos
Citotoxinas/química , Citotoxinas/metabolismo , Invertebrados/química , Animais , Cristalografia por Raios X , Microscopia de Força Atômica , Porosidade , Estrutura Secundária de Proteína , Toxinas Biológicas/químicaRESUMO
MspA has been identified as a promising candidate protein as a component of a nanopore-based DNA-sequencing device. However the wildtype protein must be engineered to incorporate all of the features desirable for an accurate and efficient device. In the present study we have utilized atomistic molecular dynamics to perform umbrella-sampling calculations to calculate the potential of mean force (PMF) profiles for translocation of the four DNA nucleotides through MspA. We show there is an energetic barrier to translocation of individual nucleotides through a mutant that closely resembles the wildtype protein, but not through a mutant engineered for the purpose of sequencing. Crucially we are able to quantify the change in free energy for mutating key residues. Thus providing a quantitative characterisation of the energetic impact of individual amino acid sidechains on nucleotide translocation through the pore of MspA.
Assuntos
DNA/química , Simulação de Dinâmica Molecular , Porinas/química , Ligação de Hidrogênio , Mutação , Porinas/genética , Porinas/metabolismo , Estrutura Terciária de Proteína , Análise de Sequência de DNA , TermodinâmicaRESUMO
Next generation DNA sequencing methods that utilize protein nanopores have the potential to revolutionize this area of biotechnology. While the technique is underpinned by simple physics, the wild-type protein pores do not have all of the desired properties for efficient and accurate DNA sequencing. Much of the research efforts have focused on protein nanopores, such as α-hemolysin from Staphylococcus aureus. However, the speed of DNA translocation has historically been an issue, hampered in part by incomplete knowledge of the energetics of translocation. Here we have utilized atomistic molecular dynamics simulations of nucleotide fragments in order to calculate the potential of mean force (PMF) through α-hemolysin. Our results reveal specific regions within the pore that play a key role in the interaction with DNA. In particular, charged residues such as D127 and K131 provide stabilizing interactions with the anionic DNA and therefore are likely to reduce the speed of translocation. These regions provide rational targets for pore optimization. Furthermore, we show that the energetic contributions to the protein-DNA interactions are a complex combination of electrostatics and short-range interactions, often mediated by water molecules.
Assuntos
DNA/química , Proteínas Hemolisinas/química , Simulação de Dinâmica Molecular , Termodinâmica , Pareamento de Bases , Staphylococcus aureus/química , Eletricidade Estática , Água/químicaRESUMO
Nanopores in membranes have a range of potential applications. Biomimetic design of nanopores aims to mimic key functions of biological pores within a stable template structure. Molecular dynamics simulations have been used to test whether a simple ß-barrel protein nanopore can be modified to incorporate a hydrophobic barrier to permeation. Simulations have been used to evaluate functional properties of such nanopores, using water flux as a proxy for ionic conductance. The behavior of these model pores has been characterized as a function of pore size and of the hydrophobicity of the amino acid side chains lining the narrow central constriction of the pore. Potential of mean force calculations have been used to calculate free energy landscapes for water and for ion permeation in selected models. These studies demonstrate that a hydrophobic barrier can indeed be designed into a ß-barrel protein nanopore, and that the height of the barrier can be adjusted by modifying the number of consecutive rings of hydrophobic side chains. A hydrophobic barrier prevents both water and ion permeation even though the pore is sterically unoccluded. These results both provide insights into the nature of hydrophobic gating in biological pores and channels, and furthermore demonstrate that simple design features may be computationally transplanted into ß-barrel membrane proteins to generate functionally complex nanopores.
Assuntos
Biomimética , Interações Hidrofóbicas e Hidrofílicas , Nanoporos , Modelos Moleculares , PermeabilidadeRESUMO
The dispersion and manipulation of carbon nanotubes (CNTs) are of great importance if we are to utilise the unique properties of CNTs in a range of biological, electrical and mechanical applications. Recently, a designed amphiphilic peptide helix termed nano-1 has been shown to solubilise CNTs in aqueous solution. Furthermore, the peptide is capable of assembling these coated tubes into fibres. We use a multiscale molecular dynamics approach to study the adsorption profile of nano-1 on a CNT surface. We find that nano-1 interacts with a CNT in a preferred orientation, such that its hydrophobic surface is in contact with the tube. The adsorption profile is unchanged upon increasing the number of peptides on the CNT. Interestingly, when few peptides are adsorbed onto the CNT surface we find that the secondary structure of the peptide is unstable. However, the helical secondary structure is stabilised upon increasing the number of peptides on the CNT surface. This study sheds light on the adsorption of peptides on CNTs, and may be exploitable to enhance the selective solubilisation and manipulation of CNTs.
Assuntos
Simulação de Dinâmica Molecular , Nanotubos de Carbono , Peptídeos/química , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestrutura , Peptídeos/metabolismo , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
Carbon nanotubes (CNTs) are possible nanoinjectors for the introduction of therapeutic agents into cells. To explore their interactions with a lipid bilayer membrane and to model the nanoinjection process, we used coarse-grained molecular dynamics to simulate the penetration of dipalmitoylphosphatidylcholine (DPPC) bilayers by single-walled CNTs. Lipids are extracted from a bilayer during CNT penetration and reside on both the inner and the outer tube surfaces. Lipids that interact with the CNT interior wall spread out and hence can "block" the tube. However, the degree of lipid lining of the inner surface is strongly dependent upon the tube penetration velocity, with fewer lipids extracted from the bilayer at higher rates. There is no apparent effect on bilayer integrity after CNT penetration, with the bilayer able to self-seal. Our findings reveal some of the complexities of the interactions of lipids with CNT nanoinjectors and suggest a need to further characterize the influence of, for example, CNT functionalization and cargo on lipid blocking of CNTs.
Assuntos
Bicamadas Lipídicas/química , Nanotubos de Carbono , Simulação por ComputadorRESUMO
Detergent interactions with carbon nanotubes are of potential importance in a number of bionanotechnology applications. We investigate the interaction of lysophospholipids with single-walled carbon nanotubes via coarse-grained molecular dynamics. We present compelling evidence that the mechanism of adsorption of these detergents onto a carbon nanotube is dependent upon detergent concentration. Furthermore, the chirality of the carbon nanotube influences the detergent wrapping angle for low detergent concentration. These findings advance our understanding of the mechanism of carbon nanotube solubilization via detergent molecules.