Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 137
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Acta Biomater ; 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34506975

RESUMO

Synthetic ß-hairpin antimicrobial peptides (AMPs) offer a useful source for the development of novel antimicrobial agents. ß-hairpin peptides generally consist of two side strands bridged by a reverse turn. In literature, most studies focused on the modifications of the side strands to manipulate the stability and activity of ß-hairpin peptides, and much less is known about the impact of the turn region. By designing a series of de novo ß-hairpin peptides with identical side strands but varied turns, we demonstrated that mutations of only 2 to 4 amino acids at the turn region could impart a wide range of antimicrobial profiles among synthetic ß-hairpin AMPs. BTT2-4 and BTT6 displayed selective potency against Gram-negative bacteria, with minimum inhibitory concentrations (MICs) of 4-8 µM. In contrast, BTT1 exhibited broad-spectrum activity, with MICs of 4-8 µM against both Gram-positive and Gram-negative strains. Additionally, BTT1 was potent against methicillin-resistant Staphylococcus aureus (MRSA) and colistin-resistant Enterobacterales. The antimicrobial potency of BTT1 persisted after 14 days of serial passage. Mechanistic studies revealed that interactions between lipopolysaccharide (LPS) and the peptides were critical to their membranolytic activity against the bacterial inner membrane. Aside from folding stability, we observed that a degree of conformational flexibility was required for disruptive membrane interactions. STATEMENT OF SIGNIFICANCE: By examining the significance of the turn region of ß-hairpin peptides, we present valuable knowledge to the design toolkit of novel antimicrobial peptides as alternative therapeutics to overcome antibiotic resistance. Our de novo designed synthetic peptides displayed selective activity against Gram-negative bacteria and potent activity against clinically relevant antibiotic-resistant strains (e.g. colistin-resistant Enterobacterales and methicillin-resistant Staphylococcus aureus). The bactericidal activity of our peptides was shown to be robust in the presence of proteolytic trypsin and saline, conditions that could suppress peptide activity. Our peptides were also determined to be non-cytotoxic against a human cell line.

2.
Nat Commun ; 12(1): 5044, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413298

RESUMO

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.


Assuntos
Mutação , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/genética , Ribossomos/patologia , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/patologia , Adolescente , Adulto , Animais , Fenômenos Biológicos , Células Cultivadas , Criança , Pré-Escolar , Dictyostelium , Drosophila , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células Germinativas , Humanos , Lactente , Simulação de Dinâmica Molecular , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Síndrome de Shwachman-Diamond/metabolismo , Adulto Jovem
3.
Biochemistry ; 60(27): 2153-2169, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34213308

RESUMO

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.


Assuntos
COVID-19/genética , Conformação Proteica , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Animais , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Chlorocebus aethiops , Glicosilação , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
4.
J Colloid Interface Sci ; 604: 670-679, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34280765

RESUMO

HYPOTHESIS: Carbon nanotubes (CNTs) represent a novel platform for cellular delivery of therapeutic peptides. Chemically-functionalized CNTs may enhance peptide uptake by improving their membrane targeting properties. EXPERIMENTS: Using coarse-grained (CG) molecular dynamics (MD) simulations, we investigate membrane interactions of a peptide conjugated to pristine and chemically-modified CNTs. As proof of principle, we focus on their interactions with PM2, an amphipathic stapled peptide that inhibits the E3 ubiquitin ligase HDM2 from negatively regulating the p53 tumor suppressor. CNT interaction with both simple planar lipid bilayers as well as spherical lipid vesicles was studied, the latter as a surrogate for curved cellular membranes. FINDINGS: Membrane permeation was rapid and spontaneous for both pristine and oxidized CNTs when unconjugated. This was slowed upon addition of a noncovalently attached peptide surface "sheath", which may be an effective way to slow CNT entry and avert membrane rupture. The CNT conjugates were observed to "desheath" their peptide layer at the bilayer interface upon insertion, leaving their cargo behind in the outer leaflet. This suggests that a synergy may exist to optimize CNT safety whilst enhancing the delivery efficiency of "hitchhiking" therapeutic molecules.


Assuntos
Nanotubos de Carbono , Membrana Celular , Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Peptídeos
5.
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
6.
Elife ; 102021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33554856

RESUMO

The spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface angiotensin-converting enzyme 2 (ACE2) receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat [HR]) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the prefusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Thus, protease docking sites flanking the S1/S2 cleavage site represent alternate allosteric hotspot targets for potential therapeutic development.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Sítios de Ligação , COVID-19/metabolismo , Humanos , Espectrometria de Massas/métodos , Simulação de Dinâmica Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteólise , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
7.
PLoS Pathog ; 17(2): e1009331, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33621239

RESUMO

Different strains within a dengue serotype (DENV1-4) can have smooth, or "bumpy" surface morphologies with different antigenic characteristics at average body temperature (37°C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Dengue/virologia , Vírus da Dengue/química , Vírus da Dengue/metabolismo , Epitopos/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Sorogrupo
8.
Methods ; 185: 28-38, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32526282

RESUMO

Enveloped viruses such as the flaviviruses represent a significant burden to human health around the world, with hundreds of millions of people each year affected by dengue alone. In an effort to improve our understanding of the molecular basis for the infective mechanisms of these viruses, extensive computational modelling approaches have been applied to elucidate their conformational dynamics. Multiscale protocols have been developed to simulate flavivirus envelopes in close accordance with biophysical data, in particular derived from cryo-electron microscopy, enabling high-resolution refinement of their structures and elucidation of the conformational changes associated with adaptation both to host environments and to immunological factors such as antibodies. Likewise, integrative modelling efforts combining data from biophysical experiments and from genome sequencing with chemical modification are providing unparalleled insights into the architecture of the previously unresolved nucleocapsid complex. Collectively, this work provides the basis for the future rational design of new antiviral therapeutics and vaccine development strategies targeting enveloped viruses.


Assuntos
Biologia Computacional/métodos , Flavivirus/química , Flavivirus/metabolismo , Modelos Moleculares , Envelope Viral/química , Envelope Viral/metabolismo , Biologia Computacional/tendências , Flavivirus/genética , Genômica/métodos , Humanos , Proteômica/métodos
9.
J Mol Cell Biol ; 12(12): 916-932, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33295606

RESUMO

There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.


Assuntos
COVID-19/complicações , Inflamação/etiologia , Lipopolissacarídeos/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Sítios de Ligação , COVID-19/imunologia , COVID-19/virologia , Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/imunologia , Modelos Animais de Doenças , Infecções por Bactérias Gram-Negativas/complicações , Infecções por Bactérias Gram-Negativas/imunologia , Humanos , Técnicas In Vitro , Lipídeo A/química , Lipídeo A/imunologia , Lipídeo A/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Imunológicos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Síndrome do Desconforto Respiratório/etiologia , Fatores de Risco , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
10.
Biochim Biophys Acta Gen Subj ; 1865(1): 129766, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33069831

RESUMO

BACKGROUND: Prediction of ligand binding and design of new function in enzymes is a time-consuming and expensive process. Crystallography gives the impression that proteins adopt a fixed shape, yet enzymes are functionally dynamic. Molecular dynamics offers the possibility of probing protein movement while predicting ligand binding. Accordingly, we choose the bacterial F1Fo ATP synthase ε subunit to unravel why ATP affinity by ε subunits from Bacillus subtilis and Bacillus PS3 differs ~500-fold, despite sharing identical sequences at the ATP-binding site. METHODS: We first used the Bacillus PS3 ε subunit structure to model the B. subtilis ε subunit structure and used this to explore the utility of molecular dynamics (MD) simulations to predict the influence of residues outside the ATP binding site. To verify the MD predictions, point mutants were made and ATP binding studies were employed. RESULTS: MD simulations predicted that E102 in the B. subtilis ε subunit, outside of the ATP binding site, influences ATP binding affinity. Engineering E102 to alanine or arginine revealed a ~10 or ~54 fold increase in ATP binding, respectively, confirming the MD prediction that E102 drastically influences ATP binding affinity. CONCLUSIONS: These findings reveal how MD can predict how changes in the "second shell" residues around substrate binding sites influence affinity in simple protein structures. Our results reveal why seemingly identical ε subunits in different ATP synthases have radically different ATP binding affinities. GENERAL SIGNIFICANCE: This study may lead to greater utility of molecular dynamics as a tool for protein design and exploration of protein design and function.


Assuntos
Trifosfato de Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Bacillus subtilis/química , Proteínas de Bactérias/química , Sítios de Ligação , ATPases Mitocondriais Próton-Translocadoras/química , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
11.
Molecules ; 25(24)2020 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-33322154

RESUMO

The ongoing development of drug resistance in HIV continues to push for the need of alternative drug targets in inhibiting HIV. One such target is the Reverse transcriptase (RT) enzyme which is unique and critical in the viral life cycle-a rational target that is likely to have less off-target effects in humans. Serendipitously, we found two chemical scaffolds from the National Cancer Institute (NCI) Diversity Set V that inhibited HIV-1 RT catalytic activity. Computational structural analyses and subsequent experimental testing demonstrated that one of the two chemical scaffolds binds to a novel location in the HIV-1 RT p51 subunit, interacting with residue Y183, which has no known association with previously reported drug resistance. This finding supports the possibility of a novel druggable site on p51 for a new class of non-nucleoside RT inhibitors that may inhibit HIV-1 RT allosterically. Although inhibitory activity was shown experimentally to only be in the micromolar range, the scaffolds serve as a proof-of-concept of targeting the HIV RT p51 subunit, with the possibility of medical chemistry methods being applied to improve inhibitory activity towards more effective drugs.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Sequência de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Sítios de Ligação , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/enzimologia , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Terapia de Alvo Molecular , Ligação Proteica , Relação Estrutura-Atividade
12.
Biomolecules ; 10(11)2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228042

RESUMO

Peptide oligomerization dynamics affects peptide structure, activity, and pharmacodynamic properties. The thrombin C-terminal peptide, TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE), is currently in preclinical development for improved wound healing and infection prevention. It exhibits turbidity when formulated at pH 7.4, particularly at concentrations of 0.3 mM or more. We used biochemical and biophysical approaches to explore whether the peptide self-associates and forms oligomers. The peptide showed a dose-dependent increase in turbidity as well as α-helical structure at pH 7.4, a phenomenon not observed at pH 5.0. By analyzing the intrinsic tryptophan fluorescence, we demonstrate that TCP-25 is more stable at high concentrations (0.3 mM) when exposed to high temperatures or a high concentration of denaturant agents, which is compatible with oligomer formation. The denaturation process was reversible above 100 µM of peptide. Dynamic light scattering demonstrated that TCP-25 oligomerization is sensitive to changes in pH, time, and temperature. Computational modeling with an active 18-mer region of TCP-25 showed that the peptide can form pH-dependent higher-order end-to-end oligomers and micelle-like structures, which is in agreement with the experimental data. Thus, TCP-25 exhibits pH- and temperature-dependent dynamic changes involving helical induction and reversible oligomerization, which explains the observed turbidity of the pharmacologically developed formulation.

13.
Sci Rep ; 10(1): 17501, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060756

RESUMO

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intent of engineering BMC-based metabolic modules for new functions in synthetic biology.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono/química , Organelas/metabolismo , Dióxido de Carbono/química , Simulação por Computador , Citoplasma/metabolismo , Ácidos Glicéricos/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Permeabilidade , Domínios Proteicos , Ribulose-Bifosfato Carboxilase/química , Eletricidade Estática , Synechococcus/metabolismo , Biologia Sintética
14.
J Chem Phys ; 153(4): 044122, 2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32752683

RESUMO

The outer membrane of Gram-negative bacteria is almost exclusively composed of lipopolysaccharide in its outer leaflet, whereas the inner leaflet contains a mixture of phospholipids. Lipopolysaccharide diffuses at least an order of magnitude slower than phospholipids, which can cause issues for molecular dynamics simulations in terms of adequate sampling. Here, we test a number of simulation protocols for their ability to achieve convergence with reasonable computational effort using the MARTINI coarse-grained force-field. This is tested in the context both of potential of mean force (PMF) calculations for lipid extraction from membranes and of lateral mixing within the membrane phase. We find that decoupling the cations that cross-link the lipopolysaccharide headgroups from the extracted lipid during PMF calculations is the best approach to achieve convergence comparable to that for phospholipid extraction. We also show that lateral lipopolysaccharide mixing/sorting is very slow and not readily addressable even with Hamiltonian replica exchange. We discuss why more sorting may be unrealistic for the short (microseconds) timescales we simulate and provide an outlook for future studies of lipopolysaccharide-containing membranes.


Assuntos
Membrana Externa Bacteriana/química , Lipídeos/isolamento & purificação , Bactérias Gram-Negativas/química , Lipídeos/química , Lipopolissacarídeos/química , Simulação de Dinâmica Molecular
15.
J Chem Theory Comput ; 16(9): 5948-5959, 2020 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-32786908

RESUMO

Molecular dynamics (MD) simulations in combination with small organic probes present in the solvent have previously been used as a method to reveal cryptic pockets that may not have been identified in experimental structures. We report such a method implemented within the CHARMM force field using the GROMACS simulation package to effectively explore cryptic pockets on the surfaces of membrane-embedded proteins using benzene as a probe molecule. This method, for which we have made implementation files freely available, relies on modified nonbonded parameters in addition to repulsive potentials between membrane lipids and benzene molecules. The method was tested on part of the outer shell of the dengue virus (DENV), for which research into a safe and effective neutralizing antibody or drug molecule is still ongoing. In particular, the envelope (E) protein, associated with the membrane (M) protein, is a lipid membrane-embedded complex which forms a dimer in the mature viral envelope. Solvent mapping was performed for the full, membrane-embedded EM protein complex and compared with similar calculations performed for the isolated, soluble E protein ectodomain dimer in the solvent. Ectodomain-only simulations with benzene exhibited unfolding effects not observed in the more physiologically relevant membrane-associated systems. A cryptic pocket which has been experimentally shown to bind n-octyl-ß-d-glucoside detergent was consistently revealed in all benzene-containing simulations. The addition of benzene also enhanced the flexibility and hydrophobic exposure of cryptic pockets at a key, functional interface in the E protein and revealed a novel, potentially druggable pocket that may be targeted to prevent conformational changes associated with viral entry into the cell.


Assuntos
Benzeno/química , Proteínas de Membrana/química , Sítios de Ligação , Vírus da Dengue/metabolismo , Dimerização , Glucosídeos/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
16.
J Phys Chem B ; 124(33): 7176-7183, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32687713

RESUMO

The membrane-embedded domain of ATP synthases contains the c-ring, which translocates ions across the membrane, and its resultant rotation is coupled to ATP synthesis in the extramembranous domain. During rotation, the c-ring becomes accessible on both sides of the lipid bilayer to solvent via channels connected to the other membrane-embedded component, the a subunit, and thereby allows the ion to be released into the solvent environment. In recent times, many experimental structures of c-rings from different species have been solved. In some of these, a water molecule with a proposed "structural role" has been identified within the c-ring ion binding site, but in general, the requirement for high resolution to resolve specific water densities complicates their interpretation. In the present study, we use molecular dynamics (MD) simulations and rigorous free energy calculations to characterize the dynamics and energetics of a water molecule within the ion binding site of the c-ring from Bacillus pseudofirmus OF4, in its wild type (WT) and P51A mutant forms, along with the c-ring from thermophilic Bacillus PS3. Our data suggest that a water molecule stably binds to the P51A mutant, as well as helping to identify a bound water molecule in Bacillus PS3 whose presence was previously overlooked due to the limited resolution of the structural data. Sequence analysis further identifies a novel conserved sequence motif that is likely required to harbor a water molecule for stable ion coordination in the binding site of such proteins.


Assuntos
Bacillus , Prótons , Trifosfato de Adenosina , Sítios de Ligação , ATPases Translocadoras de Prótons
17.
Chembiochem ; 21(22): 3249-3254, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-32608105

RESUMO

The ϵ subunit of ATP synthases has been proposed to regulate ATP hydrolysis in bacteria. Prevailing evidence supports the notion that when the ATP concentration falls below a certain threshold, the ϵ subunit changes its conformation from a non-inhibitory down-state to an extended up-state that then inhibits enzymatic ATP hydrolysis by binding to the catalytic domain. It has been demonstrated that the ϵ subunit from Bacillus PS3 is selective for ATP over other nucleotides, including GTP. In this study, the purine triphosphate selectivity is rationalized by using results from MD simulations and free energy calculations for the R103A/R115A mutant of the ϵ subunit from Bacillus PS3, which binds ATP more strongly than the wild-type protein. Our results are in good agreement with experimental data, and the elucidated molecular basis for selectivity could help to guide the design of novel GTP sensors.

18.
J Chem Inf Model ; 60(8): 3864-3883, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32702979

RESUMO

Glycans play a vital role in a large number of cellular processes. Their complex and flexible nature hampers structure-function studies using experimental techniques. Molecular dynamics (MD) simulations can help in understanding dynamic aspects of glycans if the force field parameters used can reproduce key experimentally observed properties. Here, we present optimized coarse-grained (CG) Martini force field parameters for N-glycans, calibrated against experimentally derived binding affinities for lectins. The CG bonded parameters were obtained from atomistic (ATM) simulations for different glycan topologies including high mannose and complex glycans with various branching patterns. In the CG model, additional elastic networks are shown to improve maintenance of the overall conformational distribution. Solvation free energies and octanol-water partition coefficients were also calculated for various N-glycan disaccharide combinations. When using standard Martini nonbonded parameters, we observed that glycans spontaneously aggregated in the solution and required down-scaling of their interactions for reproduction of ATM model radial distribution functions. We also optimized the nonbonded interactions for glycans interacting with seven lectin candidates and show that a relatively modest scaling down of the glycan-protein interactions can reproduce free energies obtained from experimental studies. These parameters should be of use in studying the role of glycans in various glycoproteins and carbohydrate binding proteins as well as their complexes, while benefiting from the efficiency of CG sampling.


Assuntos
Simulação de Dinâmica Molecular , Água , Polissacarídeos , Termodinâmica
19.
Biochem Biophys Res Commun ; 527(2): 518-524, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32423799

RESUMO

The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α3:ß3:γ:δ:ε:a:b2:(c2/3)9:c1 represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c1, c2 and c3, and an extra loop (γ195-211) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F1 and FO domains distinctively bended to adopt multiple conformations. Moreover, both the F1 and FO domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ195-211-loop.


Assuntos
Acetobacterium/enzimologia , Proteínas de Bactérias/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Acetobacterium/química , Acetobacterium/ultraestrutura , Proteínas de Bactérias/análise , Imageamento Tridimensional , Microscopia Eletrônica , ATPases Mitocondriais Próton-Translocadoras/análise , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/ultraestrutura
20.
Chemphyschem ; 21(9): 916-926, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32128947

RESUMO

The biochemical functions of proteins are activated at the protein glass transition temperature, which has been proposed to be dependent upon protein-water interactions. However, at the molecular level it is unclear how ligand binding to well-defined binding sites can influence this transition temperature. We thus report molecular dynamics (MD) simulations of the ϵ subunit from thermophilic Bacillus PS3 in the ATP-free and ligand-bound states over a range of temperatures from 20 to 300 K, to study the influence of ligand association upon the transition temperature. We also measure the protein mean square displacement (MSD) in each state, which is well established as a means to quantify this dynamical temperature dependence. We find that the transition temperature is largely unaffected by ligand association, but the MSD beyond the transition temperature increases more rapidly in the ATP-free state. Our data suggests that ligands can effectively "shield" a binding site from solvent, and hence stabilize protein domains with increasing temperature.


Assuntos
Proteínas/química , Temperatura de Transição , Sítios de Ligação , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Solventes/química , Termodinâmica , Água/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...