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1.
Chemistry ; 26(1): 198-205, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31643112

RESUMO

A 2D supramolecular organic framework (SOF) based on synthetic macrocycles has been constructed in water by a self-assembly strategy. Two new organic monomers of this SOF, possessing viologen and azobenzene functional groups, form a stimuli-responsive host-guest system upon cooperatively binding with cucurbit[8]uril rings. The reversible formation and dissociation of 2D SOF can be realized by the isomerization of azobenzene under ultraviolet and visible light. The light-responsive property of the SOF is highly reversible and stable for up to four cycles. Moreover, azoreductase produced by Escherichia coli can reduce the N=N double bond of azobenzene entities, resulting in fluorescence recovery of the system. As an excellent and effective fluorescent probe, the SOF can detect azoreductase activity for real-time monitoring of the growth process of Escherichia coli. The dual-stimuli responsive 2D SOF is envisioned to drive the development of responsive devices with complex functions.


Assuntos
Substâncias Macromoleculares/química , NADH NADPH Oxirredutases/metabolismo , Compostos Azo/química , Hidrocarbonetos Aromáticos com Pontes/química , Escherichia coli/metabolismo , Imidazóis/química , Isomerismo , Luz , Substâncias Macromoleculares/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Espectrometria de Fluorescência
2.
Chem Commun (Camb) ; 55(95): 14387-14390, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31723950

RESUMO

We designed a tandem stimuli-responsive assembly based on a guanidinium-modified calix[5]arene (GC5A-6C) and eosin Y modified hyaluronic acid (EY-HA), which showed hyaluronidase-triggered disassembly and ATP-activated release of EY. Both hyaluronidase and ATP are tumor biomarkers, and therefore, the present system shows potential in precision delivery with respect to tumor phototheranostics.


Assuntos
Trifosfato de Adenosina/metabolismo , Calixarenos/metabolismo , Amarelo de Eosina-(YS)/metabolismo , Guanidina/metabolismo , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Trifosfato de Adenosina/química , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Calixarenos/química , Amarelo de Eosina-(YS)/química , Guanidina/química , Humanos , Ácido Hialurônico/química , Hialuronoglucosaminidase/química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Polímeros/química , Polímeros/metabolismo , Nanomedicina Teranóstica , Microambiente Tumoral
3.
Int J Mol Sci ; 20(18)2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31533258

RESUMO

Eukaryotic genes are packed into a dynamic but stable nucleoprotein structure called chromatin. Chromatin-remodeling and modifying complexes generate a dynamic chromatin environment that ensures appropriate DNA processing and metabolism in various processes such as gene expression, as well as DNA replication, repair, and recombination. The INO80 and SWR1 chromatin remodeling complexes (INO80-c and SWR1-c) are ATP-dependent complexes that modulate the incorporation of the histone variant H2A.Z into nucleosomes, which is a critical step in eukaryotic gene regulation. Although SWR1-c has been identified in plants, plant INO80-c has not been successfully isolated and characterized. In this review, we will focus on the functions of the SWR1-c and putative INO80-c (SWR1/INO80-c) multi-subunits and multifunctional complexes in Arabidopsis thaliana. We will describe the subunit compositions of the SWR1/INO80-c and the recent findings from the standpoint of each subunit and discuss their involvement in regulating development and environmental responses in Arabidopsis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Substâncias Macromoleculares/metabolismo , Plantas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Reparo do DNA , Replicação do DNA , Histonas/metabolismo , MicroRNAs/genética , Desenvolvimento Vegetal , Imunidade Vegetal , Plantas/genética
4.
Nat Commun ; 10(1): 4347, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554812

RESUMO

Spatiotemporal control over engineered tissues is highly desirable for various biomedical applications as it emulates the dynamic behavior of natural tissues. Current spatiotemporal biomaterial functionalization approaches are based on cytotoxic, technically challenging, or non-scalable chemistries, which has hampered their widespread usage. Here we report a strategy to spatiotemporally functionalize (bio)materials based on competitive supramolecular complexation of avidin and biotin analogs. Specifically, an injectable hydrogel is orthogonally post-functionalized with desthiobiotinylated moieties using multivalent neutravidin. In situ exchange of desthiobiotin by biotin enables spatiotemporal material functionalization as demonstrated by the formation of long-range, conformal, and contra-directional biochemical gradients within complex-shaped 3D hydrogels. Temporal control over engineered tissue biochemistry is further demonstrated by timed presentation and sequestration of growth factors using desthiobiotinylated antibodies. The method's universality is confirmed by modifying hydrogels with biotinylated fluorophores, peptides, nanoparticles, enzymes, and antibodies. Overall, this work provides a facile, cytocompatible, and universal strategy to spatiotemporally functionalize materials.


Assuntos
Avidina/química , Materiais Biocompatíveis/química , Biotina/química , Substâncias Macromoleculares/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Avidina/metabolismo , Materiais Biocompatíveis/metabolismo , Biotina/análogos & derivados , Biotina/metabolismo , Biotinilação/métodos , Linhagem Celular , Humanos , Hidrogéis/química , Hidrogéis/metabolismo , Substâncias Macromoleculares/metabolismo , Camundongos , Nanopartículas/química , Peptídeos/química , Peptídeos/metabolismo , Análise Espaço-Temporal , Engenharia Tecidual/métodos
5.
Adv Virus Res ; 105: 189-238, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31522705

RESUMO

Over the last 20 years, mass spectrometry (MS), with its ability to analyze small sample amounts with high speed and sensitivity, has more and more entered the field of structural virology, aiming to investigate the structure and dynamics of viral proteins as close to their native environment as possible. The use of non-perturbing labels in hydrogen-deuterium exchange MS allows for the analysis of interactions between viral proteins and host cell factors as well as their dynamic responses to the environment. Cross-linking MS, on the other hand, can analyze interactions in viral protein complexes and identify virus-host interactions in cells. Native MS allows transferring viral proteins, complexes and capsids into the gas phase and has broken boundaries to overcome size limitations, so that now even the analysis of intact virions is possible. Different MS approaches not only inform about size, stability, interactions and dynamics of virus assemblies, but also bridge the gap to other biophysical techniques, providing valuable constraints for integrative structural modeling of viral complex assemblies that are often inaccessible by single technique approaches. In this review, recent advances are highlighted, clearly showing that structural MS approaches in virology are moving towards systems biology and ever more experiments are performed on cellular level.


Assuntos
Capsídeo/química , Capsídeo/metabolismo , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Virais/química , Proteínas Virais/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo
6.
Elife ; 82019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31552823

RESUMO

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human ß2-microglobulin (ß2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-ß structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.


Assuntos
Amiloide/química , Amiloide/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Multimerização Proteica , Microglobulina beta-2/química , Microglobulina beta-2/metabolismo , Fenômenos Biofísicos , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Ligação Proteica
7.
Nat Commun ; 10(1): 4058, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492866

RESUMO

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.


Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Substâncias Macromoleculares/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/química , DNA/ultraestrutura , DNA Helicases/química , DNA Helicases/genética , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Recombinação Homóloga , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica , Mutação , Ligação Proteica , Rad51 Recombinase/química , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
8.
Nat Struct Mol Biol ; 26(8): 744-754, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31384063

RESUMO

Precise nucleosome organization at eukaryotic promoters is thought to be generated by multiple chromatin remodeler (CR) enzymes and to affect transcription initiation. Using an integrated analysis of chromatin remodeler binding and nucleosome occupancy following rapid remodeler depletion, we investigated the interplay between these enzymes and their impact on transcription in yeast. We show that many promoters are affected by multiple CRs that operate in concert or in opposition to position the key transcription start site (TSS)-associated +1 nucleosome. We also show that nucleosome movement after CR inactivation usually results from the activity of another CR and that in the absence of any remodeling activity, +1 nucleosomes largely maintain their positions. Finally, we present functional assays suggesting that +1 nucleosome positioning often reflects a trade-off between maximizing RNA polymerase recruitment and minimizing transcription initiation at incorrect sites. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to transcription initiation.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Saccharomyces cerevisiae/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética/fisiologia , Montagem e Desmontagem da Cromatina/genética , DNA Fúngico/genética , DNA Intergênico/genética , DNA Intergênico/metabolismo , Substâncias Macromoleculares/metabolismo , Nuclease do Micrococo/metabolismo , Nucleossomos/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Colloids Surf B Biointerfaces ; 182: 110399, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31377609

RESUMO

The interaction between calf thymus DNA, ctDNA, and a series of oligomeric surfactants derived from N-benzyl-N,N-dimethyl-N-(1-dodecyl)ammonium chloride is investigated. The influence of the surfactants' degree of oligomerization (2, 3 and 4) on the ctDNA/surfactant interaction is studied, as well as the effect of the structure of the spacer group linking the individual surfactant fragments. In particular, the effect of the distance between the positive charges and the hydrophobic chains within the oligomers on these interactions was examined, by using the three positional isomers (i.e., ortho-, meta-, and para-) with the rigid xylidene moiety as spacer. Results show that the dimeric ("gemini") surfactants are much more efficient in the inversion of the nucleic acid charge than the single-chained (monomeric) surfactant. Whereas the ortho - isomer causes a partial condensation, the meta - and para - isomers can completely condense ctDNA. The meta - and para - isomers of the trimeric surfactants can also completely condense the polynucleotide. In contrast, the tetrameric surfactant investigated does not change the morphology of the nucleic acid from an elongated coil into a compacted form, in spite of effectively inverting the nucleic acid's charge in their complex. Accordingly, the capacity for ctDNA compaction of oligomeric surfactants is not simply correlated to their degree of oligomerization, but depends on a complex balance of the number and relative distance of cationic charges and/or hydrophobic tails in the surfactants for effectively interacting with the nucleic acid to form the appropriate complex. This information will help to design more effective cationic surfactants as non-viral vectors for gene therapy.


Assuntos
DNA/química , Substâncias Macromoleculares/química , Polimerização , Polímeros/química , Tensoativos/química , Cátions/química , Dicroísmo Circular , DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Substâncias Macromoleculares/metabolismo , Microscopia de Força Atômica , Estrutura Molecular , Polímeros/metabolismo , Espectrofotometria , Eletricidade Estática , Tensoativos/metabolismo
10.
Biophys Chem ; 254: 106244, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31446252

RESUMO

Macromolecules present in the intracellular environment of a cell are densely packed, resulting in a highly crowded cytosolic environment. This crowded milieu influences several biochemical equilibria such as diffusibility and association constant of biomolecules which impose a serious impact on cellular functions as well as its processes. A number of in silico and in vitro studies have been reported till date about using synthetic crowding agents for resembling such a crowding environment within the cell. Lately, it has been realized that synthetic crowders are not suitable for mimicking the intrinsic environment of the cell. In this study, proteins were assumed to be the major biological molecule which contributes to the crowding environment. We have semi-theoretically determined the total protein concentration within an individual E. coli MG1655 cell which changes notably as the growth curve proceeds from 0.2 to 1.0 OD600. The average range of total cellular protein concentration throughout the batch culture was found to be in the range of 15.2 to 178 fg/fL of cytoplasmic volume. The fundamental knowledge gained through the study was translated to applied research in the form of an equation. We propose an equation that could help to mimic the OD600 dependent crowding environment present within a single cell of E. coli in the desired volume of reaction solution. In a nutshell, the equation provides quantitative estimation of the volume of culture required to prepare the cell lysate for biomimicking the intracellular crowding environment in vitro. This finding provides a new insight into the cellular cytosolic environment that could be used as a platform to frame more cells like environment in cell-free protein synthesis (CFPS) system for synthetic biology applications.


Assuntos
Algoritmos , Escherichia coli/metabolismo , Substâncias Macromoleculares/química , Biomassa , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Substâncias Macromoleculares/metabolismo
11.
Elife ; 82019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31397671

RESUMO

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors - including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material - are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation.


Assuntos
Fatores Biológicos/metabolismo , Substâncias Macromoleculares/metabolismo , Meiose , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Microscopia de Fluorescência , Saccharomycetales/citologia , Imagem com Lapso de Tempo
12.
Nat Biomed Eng ; 3(5): 402-413, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036888

RESUMO

Cells and tissues often display pronounced spatial and dynamical metabolic heterogeneity. Common glucose-imaging techniques report glucose uptake or catabolism activity, yet do not trace the functional utilization of glucose-derived anabolic products. Here we report a microscopy technique for the optical imaging, via the spectral tracing of deuterium (STRIDE), of diverse macromolecules derived from glucose. Based on stimulated Raman-scattering imaging, STRIDE visualizes the metabolic dynamics of newly synthesized macromolecules, such as DNA, protein, lipids and glycogen, via the enrichment and distinct spectra of carbon-deuterium bonds transferred from the deuterated glucose precursor. STRIDE can also use spectral differences derived from different glucose isotopologues to visualize temporally separated glucose populations using a pulse-chase protocol. We also show that STRIDE can be used to image glucose metabolism in many mouse tissues, including tumours, brain, intestine and liver, at a detection limit of 10 mM of carbon-deuterium bonds. STRIDE provides a high-resolution and chemically informative assessment of glucose anabolic utilization.


Assuntos
Deutério/química , Glucose/metabolismo , Imagem Óptica/métodos , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Humanos , Intestinos , Lipídeos/biossíntese , Substâncias Macromoleculares/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Nus , Biossíntese de Proteínas , Análise Espectral Raman
13.
Bioresour Technol ; 287: 121406, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31103017

RESUMO

The objective of this study was to evaluate the biofixation and production of biocompounds by Chlorella fusca LEB 111 cultivated with different concentrations of carbon dioxide (CO2) adsorbent nanofibers in their free form or retained. Cultures were grown in 15% (v v-1) CO2 with 0.1, 0.3 and 0.5 g L-1 nanofibers developed with 10% (w v-1) polyacrylonitrile (PAN)/dimethylformamide (DMF), with or without nanoparticles; retained or not. The addition of 0.1 g L-1 nanofibers with nanoparticles in their free form to the cultures promoted the accumulation of approximately 3 times more carbon in the medium (46.6 mg L-1), a 45% higher biofixation rate (89.2 mg L-1 d-1) and increased carbohydrate production by approximately 2.3% (w w-1) of that observed in cultures grown without nanofibers. Therefore, nanofibers showed promising potential as physical adsorbents of CO2 in the cultivation to increase gas fixation and promote the synthesis of macromolecules.


Assuntos
Dióxido de Carbono/metabolismo , Chlorella/metabolismo , Substâncias Macromoleculares/metabolismo , Nanofibras , Carbono/metabolismo
14.
Biochemistry ; 58(17): 2208-2217, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30950267

RESUMO

In cellular environments, proteins not only interact with their specific partners but also encounter a high concentration of bystander macromolecules, or crowders. Nonspecific interactions with macromolecular crowders modulate the activities of proteins, but our knowledge about the rules of nonspecific interactions is still very limited. In previous work, we presented experimental evidence that macromolecular crowders acted competitively in inhibiting the binding of maltose binding protein (MBP) with its ligand maltose. Competition between a ligand and an inhibitor may result from binding to either the same site or different conformations of the protein. Maltose binds to the cleft between two lobes of MBP, and in a series of mutants, the affinities increased with an increase in the extent of lobe closure. Here we investigated whether macromolecular crowders also have a conformational or site preference when binding to MBP. The affinities of a polymer crowder, Ficoll70, measured by monitoring tryptophan fluorescence were 3-6-fold higher for closure mutants than for wild-type MBP. Competition between the ligand and crowder, as indicated by fitting of titration data and directly by nuclear magnetic resonance spectroscopy, and their similar preferences for closed MBP conformations further suggest the scenario in which the crowder, like maltose, preferentially binds to the interlobe cleft of MBP. Similar observations were made for bovine serum albumin as a protein crowder. Conformational and site preferences in MBP-crowder binding allude to the paradigm that nonspecific interactions can possess hallmarks of molecular recognition, which may be essential for intracellular organizations including colocalization of proteins and liquid-liquid phase separation.


Assuntos
Substâncias Macromoleculares/química , Proteínas Ligantes de Maltose/química , Conformação Proteica , Ligantes , Substâncias Macromoleculares/metabolismo , Espectroscopia de Ressonância Magnética , Maltose/química , Maltose/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica
15.
Biochemistry ; 58(15): 1963-1974, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30950607

RESUMO

A( syn)-T and G( syn)-C+ Hoogsteen base pairs in protein-bound DNA duplexes can be difficult to resolve by X-ray crystallography due to ambiguous electron density and by nuclear magnetic resonance (NMR) spectroscopy due to poor chemical shift dispersion and size limitations with solution-state NMR spectroscopy. Here we describe an NMR strategy for characterizing Hoogsteen base pairs in protein-DNA complexes, which relies on site-specifically incorporating 13C- and 15N-labeled nucleotides into DNA duplexes for unambiguous resonance assignment and to improve spectral resolution. The approach was used to resolve the conformation of an A-T base pair in a crystal structure of an ∼43 kDa complex between a 34 bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (Protein Data Bank entry 1IHF ), this base pair adopts an unusual Hoogsteen conformation with a distorted sugar backbone that is accommodated by a nearby nick used to aid in crystallization. The NMR chemical shifts and interproton nuclear Overhauser effects indicate that this base pair predominantly adopts a Watson-Crick conformation in the intact DNA-IHF complex under solution conditions. Consistent with these NMR findings, substitution of 7-deazaadenine at this base pair resulted in only a small (∼2-fold) decrease in the IHF-DNA binding affinity. The NMR strategy provides a new approach for resolving crystallographic ambiguity and more generally for studying the structure and dynamics of protein-DNA complexes in solution.


Assuntos
Pareamento de Bases , Proteínas de Ligação a DNA/química , DNA/química , Substâncias Macromoleculares/química , Espectroscopia de Ressonância Magnética/métodos , Conformação de Ácido Nucleico , Sequência de Bases , Isótopos de Carbono/metabolismo , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Substâncias Macromoleculares/metabolismo , Modelos Moleculares , Estrutura Molecular , Isótopos de Nitrogênio/metabolismo , Nucleotídeos/química , Nucleotídeos/genética , Nucleotídeos/metabolismo , Domínios Proteicos
16.
Spectrochim Acta A Mol Biomol Spectrosc ; 217: 278-287, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30952094

RESUMO

By using first-principles approaches based on Density Functional Theory, we explore the possibility of using dendritic macromolecular structures as carriers of the doxorubicin anticancer drug. In particular, we consider macromolecular cavities of different sizes composed of phenylene-, thiophene-, phenyl-cored thiophen- and thioazole-based dendrimers. The comparison between the optimized molecular geometries of the monomers and of the host-guest complexes reveals that only slight structural changes are observed in doxorubicin upon complexation. Also, the encapsulation energies for the host-guest complexes suggest that these systems are of potential use for pharmacology applications in vivo. The interaction of the guest doxorubicin with the macromolecular cavities exploits different types of weak intermolecular forces including σ, π and hydrogen bond interactions. The electronic structure of these complexes is discussed, with particular emphasis placed on the role of the charge distribution and the nature of the frontier molecular orbitals in the encapsulation process. Spectroscopic properties of these complexes are derived to facilitate their detection in laboratory and in vivo. These include IR vibrational frequencies, absorption wavelengths and relative oscillator strengths for the main transitions in the UV-Vis spectrum.


Assuntos
Antibióticos Antineoplásicos/química , Dendrímeros/química , Doxorrubicina/química , Lipossomos/química , Substâncias Macromoleculares/química , Modelos Teóricos , Antibióticos Antineoplásicos/metabolismo , Benchmarking , Cápsulas , Dendrímeros/metabolismo , Doxorrubicina/metabolismo , Lipossomos/metabolismo , Substâncias Macromoleculares/metabolismo , Teoria Quântica , Termodinâmica
17.
FEBS Open Bio ; 9(3): 420-427, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30868050

RESUMO

The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA. The uracil base is among the most frequently occurring erroneous bases in DNA, and is cut out from the phosphodiester backbone via the catalytic action of uracil-DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil-DNA glycosylase. Interestingly, both uracil-DNA glycosylase (Staphylococcus aureus uracil-DNA glycosylase; SAUDG) and its inhibitor (S. aureus uracil-DNA glycosylase inhibitor; SAUGI) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil-DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI. Here, we investigated whether these mutations drastically perturb the interaction with SAUDG. To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these K d values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil-DNA glycosylase inhibitor proteins.


Assuntos
Substâncias Macromoleculares/análise , Mutação , Staphylococcus aureus/enzimologia , Uracila-DNA Glicosidase/química , Inibidores Enzimáticos/farmacologia , Substâncias Macromoleculares/antagonistas & inibidores , Substâncias Macromoleculares/metabolismo , Espectrometria de Massas , Modelos Moleculares , Uracila-DNA Glicosidase/antagonistas & inibidores , Uracila-DNA Glicosidase/metabolismo
18.
J Bacteriol ; 201(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30833357

RESUMO

Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well.IMPORTANCE Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm.


Assuntos
Adaptação Fisiológica , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Substâncias Macromoleculares/metabolismo , Pressão Osmótica , Fenômenos Bioquímicos , Divisão Celular
19.
Molecules ; 24(5)2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857224

RESUMO

The plasma membrane H⁺-ATPase was purified from the yeast K. lactis. The oligomeric state of the H⁺-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H⁺-ATPase hexamer in both MASs as the sole/main oligomeric state-in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H⁺-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H⁺-ATPase. It is concluded that the quaternary structure of the H⁺-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.


Assuntos
Membrana Celular/enzimologia , Kluyveromyces/enzimologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Western Blotting , Cromatografia em Gel , Substâncias Macromoleculares/metabolismo
20.
Int J Mol Sci ; 20(5)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871103

RESUMO

Interaction of proteins with charged macromolecules is involved in many processes in cells. Firstly, there are many naturally occurred charged polymers such as DNA and RNA, polyphosphates, sulfated glycosaminoglycans, etc., as well as pronouncedly charged proteins such as histones or actin. Electrostatic interactions are also important for "generic" proteins, which are not generally considered as polyanions or polycations. Finally, protein behavior can be altered due to post-translational modifications such as phosphorylation, sulfation, and glycation, which change a local charge of the protein region. Herein we review molecular modeling for the investigation of such interactions, from model polyanions and polycations to unfolded proteins. We will show that electrostatic interactions are ubiquitous, and molecular dynamics simulations provide an outstanding opportunity to look inside binding and reveal the contribution of electrostatic interactions. Since a molecular dynamics simulation is only a model, we will comprehensively consider its relationship with the experimental data.


Assuntos
Substâncias Macromoleculares/metabolismo , Polímeros/metabolismo , Proteínas/metabolismo , Animais , Humanos , Simulação de Dinâmica Molecular , Poliaminas/metabolismo , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Eletricidade Estática
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