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

RESUMO

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

2.
JAC Antimicrob Resist ; 3(3): dlab118, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34396124

RESUMO

Background: There is limited literature evaluating the effect of antibiotic stewardship programmes (ASPs) in hospitalized geriatric patients, who are at higher risk for readmissions, developing Clostridioides difficile infection (CDI) or other adverse outcomes secondary to antibiotic treatments. Methods: In this cohort study we compare the rates of 30 day hospital readmissions because of reinfection or development of CDI in patients 65 years and older who received ASP interventions between January and June 2017. We also assessed their mortality rates and length of stay. Patients were included if they received antibiotics for pneumonia, urinary tract infection, acute bacterial skin and skin structure infection or complicated intra-abdominal infection. The ASP team reviewed patients on antibiotics daily. ASP interventions included de-escalation of empirical or definitive therapy, change in duration of therapy or discontinuation of therapy. Treatment failure was defined as readmission because of reinfection or a new infection. A control group of patients 65 years and older who received antibiotics between January and June 2015 (pre-ASP) was analysed for comparison. Results: We demonstrated that the 30 day hospital readmission rate for all infection types decreased during the ASP intervention period from 24.9% to 9.3%, P < 0.001. The rate of 30 day readmissions because of CDI decreased during the intervention period from 2.4% to 0.30%, P = 0.02. Mortality in the cohort that underwent ASP interventions decreased from 9.6% to 5.4%, P = 0.03. Lastly, antibiotic expenditure decreased after implementation of the ASP from $23.3 to $4.3 per adjusted patient day, in just 6 months. Conclusions: Rigorous de-escalation and curtailing of antibiotic therapies were beneficial and without risk for the hospitalized patients 65 years and over.

3.
Commun Biol ; 3(1): 766, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318620

RESUMO

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


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas , Lipídeos , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Nanoestruturas , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/metabolismo , Catálise , Complexos Multiproteicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Proteolipídeos/metabolismo
4.
Lancet Rheumatol ; 2(10): e603-e612, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32838323

RESUMO

Background: Tocilizumab, a monoclonal antibody directed against the interleukin-6 receptor, has been proposed to mitigate the cytokine storm syndrome associated with severe COVID-19. We aimed to investigate the association between tocilizumab exposure and hospital-related mortality among patients requiring intensive care unit (ICU) support for COVID-19. Methods: We did a retrospective observational cohort study at 13 hospitals within the Hackensack Meridian Health network (NJ, USA). We included patients (aged ≥18 years) with laboratory-confirmed COVID-19 who needed support in the ICU. We obtained data from a prospective observational database and compared outcomes in patients who received tocilizumab with those who did not. We applied a multivariable Cox model with propensity score matching to reduce confounding effects. The primary endpoint was hospital-related mortality. The prospective observational database is registered on ClinicalTrials.gov, NCT04347993. Findings: Between March 1 and April 22, 2020, 764 patients with COVID-19 required support in the ICU, of whom 210 (27%) received tocilizumab. Factors associated with receiving tocilizumab were patients' age, gender, renal function, and treatment location. 630 patients were included in the propensity score-matched population, of whom 210 received tocilizumab and 420 did not receive tocilizumab. 358 (57%) of 630 patients died, 102 (49%) who received tocilizumab and 256 (61%) who did not receive tocilizumab. Overall median survival from time of admission was not reached (95% CI 23 days-not reached) among patients receiving tocilizumab and was 19 days (16-26) for those who did not receive tocilizumab (hazard ratio [HR] 0·71, 95% CI 0·56-0·89; p=0·0027). In the primary multivariable Cox regression analysis with propensity matching, an association was noted between receiving tocilizumab and decreased hospital-related mortality (HR 0·64, 95% CI 0·47-0·87; p=0·0040). Similar associations with tocilizumab were noted among subgroups requiring mechanical ventilatory support and with baseline C-reactive protein of 15 mg/dL or higher. Interpretation: In this observational study, patients with COVID-19 requiring ICU support who received tocilizumab had reduced mortality. Results of ongoing randomised controlled trials are awaited. Funding: None.

5.
Nat Commun ; 11(1): 2155, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358557

RESUMO

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


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Proteínas de Transporte/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Espectrometria de Massas , Chaperonas Moleculares/genética , Peptidilprolil Isomerase/genética , Ligação Proteica
6.
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
7.
Biophys J ; 116(7): 1194-1203, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30885379

RESUMO

Hydrogen/deuterium exchange monitored by mass spectrometry is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is generally not possible. Here, we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) may be extracted and the uniqueness of the solutions obtained depending on the degree of peptide overlap. However, in most cases, the solution is not unique, and multiple alternatives must be considered. We provide a statistical method that clusters the solutions to further reduce their number. Such analysis always provides meaningful constraints on protection factors and can be used in situations in which obtaining more refined experimental data is impractical. It also provides a systematic way to improve data collection strategies to obtain unambiguous information at single-residue level (e.g., for assessing protein structure predictions at atomistic level).


Assuntos
Deutério/química , Espectrometria de Massas/métodos , Peptídeos/química , Amidas/química , Complemento C3/química , Ligação de Hidrogênio , Espectrometria de Massas/normas
8.
Mol Cell ; 74(3): 584-597.e9, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30905508

RESUMO

V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability.


Assuntos
Instabilidade Genômica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Recombinação V(D)J/genética , Animais , Sequência de Bases/genética , Células COS , Chlorocebus aethiops , Cromossomos/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Células NIH 3T3 , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Recombinases/genética
9.
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
10.
Elife ; 72018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29877797

RESUMO

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.


Assuntos
Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Força Próton-Motriz , Canais de Translocação SEC/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidrólise , Microscopia de Fluorescência/métodos , Modelos Moleculares , Mutação , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Canais de Translocação SEC/química , Canais de Translocação SEC/genética , Proteínas SecA
11.
Nucleic Acids Res ; 46(15): 7924-7937, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-29796667

RESUMO

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


Assuntos
Chaperonas Moleculares/metabolismo , Orthoreovirus Aviário/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Bases , Genoma Viral/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Orthoreovirus Aviário/genética , Ligação Proteica , Estrutura Secundária de Proteína , RNA Viral/química , RNA Viral/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
12.
Emerg Infect Dis ; 24(3): 584-587, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29460760

RESUMO

In 2015, Clostridium difficile testing rates among 30 US community, multispecialty, and cancer hospitals were 14.0, 16.3, and 33.9/1,000 patient-days, respectively. Pooled hospital onset rates were 0.56, 0.84, and 1.57/1,000 patient-days, respectively. Higher testing rates may artificially inflate reported rates of C. difficile infection. C. difficile surveillance should consider testing frequency.


Assuntos
Clostridioides difficile , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Disparidades nos Níveis de Saúde , Técnicas Bacteriológicas , Clostridioides difficile/genética , Infecções por Clostridium/diagnóstico , Hospitalização , Hospitais , Humanos , Técnicas de Amplificação de Ácido Nucleico , Vigilância em Saúde Pública
13.
Proc Natl Acad Sci U S A ; 114(46): 12255-12260, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29087310

RESUMO

Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes.


Assuntos
Proteínas do Capsídeo/química , Engenharia Genética , Genoma Viral , RNA Viral/química , Vírus Satélite da Necrose do Tabaco/genética , Montagem de Vírus , Motivos de Aminoácidos , Sítios de Ligação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Expressão Gênica , Tamanho do Genoma , Sequências Repetidas Invertidas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , RNA Viral/genética , RNA Viral/metabolismo , Vírus Satélite da Necrose do Tabaco/metabolismo , Vírus Satélite da Necrose do Tabaco/ultraestrutura , Replicação Viral
14.
Nat Microbiol ; 2: 17098, 2017 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-28628133

RESUMO

Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and the hepatitis B core protein that play roles in defining the nucleocapsid assembly pathway. Using RNA SELEX and bioinformatics, we identified multiple regions in the pre-genomic RNA with high affinity for core protein dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-electron microscopy reconstruction of the T = 4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main core protein shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of core protein. These core protein-RNA contacts may play one or more roles in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for nucleocapsid assembly.


Assuntos
Vírus da Hepatite B/fisiologia , Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Proteínas do Core Viral/metabolismo , Montagem de Vírus , Sítios de Ligação , Biologia Computacional , Ligação Proteica , Técnica de Seleção de Aptâmeros
15.
Biochim Biophys Acta Bioenerg ; 1858(9): 763-770, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28634030

RESUMO

The molecular mechanism of proton pumping by heme-copper oxidases (HCO) has intrigued the scientific community since it was first proposed. We have recently reported a novel technology that enables the continuous characterisation of proton transport activity of a HCO and ubiquinol oxidase from Escherichia coli, cytochrome bo3, for hundreds of seconds on the single enzyme level (Li et al. J Am Chem Soc 137 (2015) 16055-16063). Here, we have extended these studies by additional experiments and analyses of the proton transfer rate as a function of proteoliposome size and pH at the N- and P-side of single HCOs. Proton transport activity of cytochrome bo3 was found to decrease with increased curvature of the membrane. Furthermore, proton uptake at the N-side (proton entrance) was insensitive to pH between pH6.4-8.4, while proton release at the P-side had an optimum pH of ~7.4, suggesting that the pH optimum is related to proton release from the proton exit site. Our previous single-enzyme experiments identified rare, long-lived conformation states of cytochrome bo3 where protons leak back under turn-over conditions. Here, we analyzed and found that ~23% of cytochrome bo3 proteoliposomes show ΔpH half-lives below 50s after stopping turnover, while only ~5% of the proteoliposomes containing a non-pumping mutant, E286C cytochrome bo3 exhibit such fast decays. These single-enzyme results confirm our model in which HCO exhibit heterogeneous pumping rates and can adopt rare leak states in which protons are able to rapidly flow back.


Assuntos
Citocromos/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Proteolipídeos/metabolismo , Bombas de Próton/metabolismo , Transporte Biológico , Grupo dos Citocromos b , Citocromos/genética , Técnicas Eletroquímicas/instrumentação , Transporte de Elétrons , Escherichia coli/enzimologia , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Corantes Fluorescentes , Lipossomos/metabolismo , Microscopia de Fluorescência , Oxirredução , Proteolipídeos/ultraestrutura , Bombas de Próton/genética , Prótons
16.
Proc Natl Acad Sci U S A ; 114(18): 4673-4678, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28416674

RESUMO

Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein's structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, ß2-microglobulin (ß2m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36-1.8 ms, at concentrations as low as 0.5 mg mL-1 In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.


Assuntos
Anticorpos Monoclonais/química , Fator Estimulador de Colônias de Granulócitos/química , Hidrodinâmica , Agregados Proteicos , Soroalbumina Bovina/química , Microglobulina beta-2/química , Animais , Bovinos , Humanos , Cinética , Estabilidade Proteica
17.
Biophys J ; 111(10): 2077-2085, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27851933

RESUMO

Long RNA molecules are at the core of gene regulation across all kingdoms of life, while also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with nonrepeating sequences usually adopt highly ramified secondary structures and are better described as branched polymers. To test whether a branched polymer model can estimate the overall sizes of large RNAs, we employed fluorescence correlation spectroscopy to examine the hydrodynamic radii of a broad spectrum of biologically important RNAs, ranging from viral genomes to long noncoding regulatory RNAs. The relative sizes of long RNAs measured at low ionic strength correspond well to those predicted by two theoretical approaches that treat the effective branching associated with secondary structure formation-one employing the Kramers theorem for calculating radii of gyration, and the other featuring the metric of maximum ladder distance. Upon addition of multivalent cations, most RNAs are found to be compacted as compared with their original, low ionic-strength sizes. These results suggest that sizes of long RNA molecules are determined by the branching pattern of their secondary structures. We also experimentally validate the proposed computational approaches for estimating hydrodynamic radii of single-stranded RNAs, which use generic RNA structure prediction tools and thus can be universally applied to a wide range of long RNAs.


Assuntos
Conformação de Ácido Nucleico , RNA/química , Sequência de Bases , Hidrodinâmica , Modelos Moleculares , RNA/genética
18.
Methods Mol Biol ; 1431: 175-93, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27283310

RESUMO

Chromatin-remodeling ATPases modulate histones-DNA interactions within nucleosomes and regulate transcription. At the heart of remodeling, ATPase is a helicase-like motor flanked by a variety of conserved targeting domains. CHD4 is the core subunit of the nucleosome remodeling and deacetylase complex NuRD and harbors tandem plant homeo finger (tPHD) and chromo (tCHD) domains. We describe a multifaceted approach to link the domain structure with function, using quantitative assays for DNA and histone binding, ATPase activity, shape reconstruction from solution scattering data, and single molecule translocation assays. These approaches are complementary to high-resolution structure determination.


Assuntos
Autoantígenos/química , Autoantígenos/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Fenômenos Biofísicos , DNA/metabolismo , Difusão Dinâmica da Luz , Histonas/metabolismo , Humanos , Domínios Proteicos , Transporte Proteico , Imagem Individual de Molécula
19.
Elife ; 52016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27183269

RESUMO

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Escherichia coli/metabolismo , Methanocaldococcus/metabolismo , Canais de Translocação SEC/química , Thermotoga maritima/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Expressão Gênica , Cinética , Methanocaldococcus/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Proteínas SecA , Especificidade por Substrato , Termodinâmica , Thermotoga maritima/genética
20.
Bacteriophage ; 6(1): e1157666, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27144089

RESUMO

Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These show remarkable overlap with previous predictions based on the hypothesis that virion assembly is mediated by multiple sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the dominant assembly pathway and ensure cognate RNA encapsidation. In MS2, they also trigger conformational change in the dimeric capsomere creating the A/B quasi-conformer, 60 of which are needed to complete the T=3 capsid. This is the most compelling demonstration to date that this ssRNA virus, and by implications potentially very many of them, assemble via a PS-mediated assembly mechanism.

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