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1.
Phys Chem Chem Phys ; 22(3): 1611-1623, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31894790

RESUMO

Electronic circular dichroism is one of the most used spectroscopic techniques for peptide and protein structural characterization. However, while valuable experimental spectra exist for α-helix, ß-sheet and random coil secondary structures, previous studies showed important discrepancies for ß-turns, limiting their use as a reference for structural studies. In this paper, we simulated circular dichroism spectra for the best-characterized ß-turns in peptides, namely types I, II, I' and II'. In particular, by combining classical molecular dynamics simulations and state-of-the-art quantum time-dependent density functional theory (with the polarizable embedding multiscale model) computations, two common electronic circular dichroism patterns were found for couples of ß-turn types (namely, type I/type II' and type II/type I'), at first for a minimal di-peptide model (Ace-Ala-Ala-NHMe), but also for all sequences tested with non-aromatic residues in the central positions. On the other hand, as expected, aromatic substitution causes important perturbations to the previously found ECD patterns. Finally, by applying suitable approximations, these patterns were subsequently rationalized based on the exciton chirality rule. All these results provide useful predictions and pave the way for a possible experimental characterization of ß-turns based on circular dichroism spectroscopy.


Assuntos
Dicroísmo Circular , Simulação de Dinâmica Molecular , Simulação por Computador , Conformação Proteica em Folha beta , Estrutura Terciária de Proteína
2.
Phys Chem Chem Phys ; 22(5): 2938-2948, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31951234

RESUMO

Eukaryotic translation initiation factor 4E binding protein 2 (4E-BP2) is an inhibitor of mRNA cap-dependent translations. Wild-type (WT) 4E-BP2 is intrinsically disordered under physiological conditions, while phosphorylation converts the disordered fragments 18-62 into a four-stranded ß-sheet structure. The regulation mechanism of phosphorylation on 4E-BP2 still remains ambiguous. In this study, replica-exchange molecular dynamics (REMD) simulations were utilized to sample the conformation spaces of WT, phosphorylated WT (pWT), and phosphorylated mutated (pMT) 4E-BP2. Starting from extended structures, the folded structures were only observed in pWT simulations. The folding pathway shows that the folded structures of pWT are formed in the order of ß1/ß4, ß3, and ß2. The formation of ß-turns on pWT, which are driven by hydrogen bonds between the phosphorylated residues and adjacent residues, are the rate-limiting steps in the folding process. The long-range electrostatic interactions contribute toward the stabilization of the folded structures. Moreover, the disruption of ß-turn structures induced by mutations would prevent the folding of pMT 4E-BP2. Our finding is helpful in understanding the regulation of the structural ensembles of intrinsically disordered proteins.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Sequência de Aminoácidos , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Humanos , Ligações de Hidrogênio , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Conformação Proteica em Folha beta , Dobramento de Proteína , Termodinâmica
3.
Phys Chem Chem Phys ; 22(3): 1359-1370, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31854397

RESUMO

Depending on the amino acid sequence, as well as the local environment, some peptides have the capability to fold into multiple secondary structures. Conformational switching between such structures is a key element of protein folding and aggregation. Specifically, understanding the molecular mechanism underlying the transition from an α-helix to a ß-hairpin is critical because it is thought to be a harbinger of amyloid assembly. In this study, we explore the energy landscape for an 18-residue peptide (DP5), designed by Araki and Tamura to exhibit equal propensities for the α-helical and ß-hairpin forms. We find that the degeneracy is encoded in the multifunnel nature of the underlying free energy landscape. In agreement with experiment, we also observe that mutation of tyrosine at position 12 to serine shifts the equilibrium in favor of the α-helix conformation, by altering the landscape topography. The transition from the α-helix to the ß-hairpin is a complex stepwise process, and occurs via collapsed coil-like intermediates. Our findings suggest that even a single mutation can tune the emergent features of the landscape, providing an efficient route to protein design. Interestingly, the transition pathways for the conformational switch seem to be minimally perturbed upon mutation, suggesting that there could be universal microscopic features that are conserved among different switch-competent protein sequences.


Assuntos
Peptídeos/química , Mutação , Peptídeos/genética , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética
4.
Food Chem ; 309: 125651, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31678674

RESUMO

The effects of low frequency magnetic field on myoglobin (Mb) oxidation stability were evaluated by treatments at 0, 3, 6, 9, 12 mT and storage for 10 h. The results showed that Mb oxidation was inhibited under all magnetic field treatments, due to the increase of total sulfhydryl and free amino groups (9 or 12 mT) from unfolding of Mb clusters (3, 9, 12 mT) as well as ß-turn and ß-sheet structures (9 or 12 mT). The unfolding also induced (i) the destruction or burial of iron porphyrin and tyrosine residues; (ii) the exposure of tryptophan; (iii) more uniform Mb particle size distribution (3, 9, 12 mT) and increased zeta potential (3, 6, 12 mT). Overall, magnetic field promoted exposed active groups as the preferred oxidation target, thus decreasing the oxidation rate of central iron atoms. It also promoted Mb stability by redistributing particle size and increasing zeta potential.


Assuntos
Mioglobina/química , Aminas/análise , Campos Magnéticos , Oxirredução , Tamanho da Partícula , Conformação Proteica em Folha beta , Estabilidade Proteica , Desdobramento de Proteína , Compostos de Sulfidrila/análise
5.
Food Chem ; 309: 125757, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31699562

RESUMO

Impacts of superfine grinding treatment (0, 2, 4, 6, 8 or 10 h) on structure, physicochemical and rheological properties of transglutaminase (TGase)-crosslinked whey protein isolate (WPI) were investigated. Size exclusion chromatography showed that high molecular weight polymers were formed in TGase-treated sWPI (WPI treated with superfine grinding), whereas its consumption of free amino groups reached the maximum at grinding 8 h and 10 h. With the milling time extended from 0 to 10 h, particle size of the TGase-crosslinked sWPI gradually increased. Emission spectrum shifted from 338.6 nm to 340.6 nm after sWPI treated with TGase. Meanwhile, TGase-crosslinked sWPI had higher apparent viscosity, emulsifying properties than TGase-crosslinked WPI, but the solubility of TGase-crosslinked sWPI was lower than sWPI. Superfine grinding treatment remarkably increased ß-sheet and random compositions in TGase-crosslinked sWPI. These findings indicated that superfine grinding treatment could enhance the TGase cross-linking degree, and improve rheological properties in TGase-crosslinked WPI.


Assuntos
Transglutaminases/metabolismo , Proteínas do Soro do Leite/metabolismo , Emulsões/química , Peso Molecular , Tamanho da Partícula , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Reologia , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Viscosidade , Proteínas do Soro do Leite/química
6.
Biochemistry (Mosc) ; 84(11): 1256-1267, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760916

RESUMO

The review discusses the role of small heat shock proteins (sHsps) in human neurodegenerative disorders, such as Charcot-Marie-Tooth disease (CMT), Parkinson's and Alzheimer's diseases, and different forms of tauopathies. The effects of CMT-associated mutations in two small heat shock proteins (HspB1 and HspB8) on the protein stability, oligomeric structure, and chaperone-like activity are described. Mutations in HspB1 shift the equilibrium between different protein oligomeric forms, leading to the alterations in its chaperone-like activity and interaction with protein partners, which can induce damage of the cytoskeleton and neuronal death. Mutations in HspB8 affect its interaction with the adapter protein Bag3, as well as the process of autophagy, also resulting in neuronal death. The impact of sHsps on different forms of amyloidosis is discussed. Experimental studies have shown that sHsps interact with monomers or small oligomers of amyloidogenic proteins, stabilize their structure, prevent their aggregation, and/or promote their specific proteolytic degradation. This effect might be due to the interaction between the ß-strands of sHsps and ß-strands of target proteins, which prevents aggregation of the latter. In cooperation with the other heat shock proteins, sHsps can promote disassembly of oligomers formed by amyloidogenic proteins. Despite significant achievements, further investigations are required for understanding the role of sHsps in protection against various neurodegenerative diseases.


Assuntos
Proteínas de Choque Térmico Pequenas/metabolismo , Doenças Neurodegenerativas/patologia , Amiloidose/metabolismo , Amiloidose/patologia , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/genética , Humanos , Doenças Neurodegenerativas/metabolismo , Conformação Proteica em Folha beta , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
7.
Phys Chem Chem Phys ; 21(44): 24601-24619, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31670335

RESUMO

Natural bond orbital (NBO) analysis of electron delocalization in a series of capped isolated peptides is used to diagnose amide-amide H-bonding and backbone-induced hyperconjugative interactions, and to rationalize their spectral effects. The sum of the stabilization energies corresponding to the interactions between NBOs that are involved in the H-bonding is demonstrated as an insightful indicator for the H-bond strength. It is then used to decouple the effect of the H-bond distance from that, intrinsic, of the donor/acceptor relative orientation, i.e., the geometrical approach. The diversity of the approaches given by the series of peptides studied enables us to illustrate the crucial importance of the approach when the acceptor is a carbonyl group, and emphasizes that efficient approaches can be achieved despite not matching the usual picture of a proton donor directly facing a lone pair of the proton acceptor, i.e., that encountered in intermolecular H-bonds. The study also illustrates the role of backbone flexibility, partly controlled by backbone-amide hyperconjugative interactions, in influencing the equilibrium structures, in particular by frustrating or enhancing the HB for a given geometrical approach. Finally, the presently used NBO-based HB strength indicator enables a fair prediction of the frequency of the proton donor amide NH stretching mode, but this simple picture is blurred by ubiquitous hyperconjugative effects between the backbone and amide groups, whose magnitude can be comparable to that of the weakest H-bonds.


Assuntos
Amidas/química , Peptídeos/química , Dimerização , Ligações de Hidrogênio , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Teoria Quântica
8.
Nucleic Acids Res ; 47(16): 8874-8887, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616952

RESUMO

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein-protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis ß-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.


Assuntos
Bacteriófago lambda/genética , Cromossomos Bacterianos/química , DNA Nucleotidiltransferases/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/química , Proteínas Virais/química , Sítio Alostérico , Bacteriófago lambda/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/genética , Fator Proteico para Inversão de Estimulação/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparo de DNA por Recombinação , Alinhamento de Sequência , Termodinâmica , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
Phys Chem Chem Phys ; 21(44): 24393-24405, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31663524

RESUMO

Engineered repeat proteins have proven to be a fertile ground for studying the competition between folding, misfolding and transient aggregation of tethered protein domains. We examine the interplay between folding and inter-domain interactions of engineered FiP35 WW domain repeat proteins with n = 1 through 5 repeats. We characterize protein expression, thermal and guanidium melts, as well as laser T-jump kinetics. All experimental data is fitted by a global fitting model with two states per domain (U, N), plus a third state M to account for non-native states due to domain interactions present in all but the monomer. A detailed structural model is provided by coarse-grained simulated annealing using the AWSEM Hamiltonian. Tethered FiP35 WW domains with n = 2 and 3 domains are just slightly less stable than the monomer. The n = 4 oligomer is yet less stable, its expression yield is much lower than the monomer's, and depends on the purification tag used. The n = 5 plasmid did not express at all, indicating the sudden onset of aggregation past n = 4. Thus, tethered FiP35 has a critical nucleus size for inter-domain aggregation of n ≈ 4. According to our simulations, misfolded structures become increasingly prevalent as one proceeds from monomer to pentamer, with extended inter-domain beta sheets appearing first, then multi-sheet 'intramolecular amyloid' structures, and finally novel motifs containing alpha helices. We discuss the implications of our results for oligomeric aggregate formation and structure, transient aggregation of proteins whilst folding, as well as for protein evolution that starts with repeat proteins.


Assuntos
Proteínas/química , Cinética , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Proteínas/genética , Proteínas/metabolismo , Termodinâmica , Domínios WW
10.
Nat Commun ; 10(1): 4708, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31624265

RESUMO

Self-assembling peptides have the ability to spontaneously aggregate into large ordered structures. The reversibility of the peptide hydrogen bonded supramolecular assembly make them tunable to a host of different applications, although it leaves them highly dynamic and prone to disassembly at the low concentration needed for biological applications. Here we demonstrate that a secondary hydrophobic interaction, near the peptide core, can stabilise the highly dynamic peptide bonds, without losing the vital solubility of the systems in aqueous conditions. This hierarchical self-assembly process can be used to stabilise a range of different ß-sheet hydrogen bonded architectures.


Assuntos
Substâncias Macromoleculares/química , Nanotubos de Peptídeos/química , Peptídeos/química , Conformação Proteica em Folha beta , Água/química , Sobrevivência Celular , Humanos , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Células PC-3 , Solubilidade , Termodinâmica
11.
J Chem Phys ; 151(10): 105101, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31521094

RESUMO

The diffusion map is a dimensionality reduction method. The reduction coordinates are associated with the leading eigenfunctions of the backward Fokker-Planck operator, providing a dynamic meaning for these coordinates. One of the key factors that affect the accuracy of diffusion map embedding is the dynamic measure implemented in the Gaussian kernel. A common practice in diffusion map study of molecular systems is to approximate dynamic proximity with RMSD (root-mean-square deviation). In this paper, we present a hybrid geometry-energy based kernel. Since high energy-barriers may exist between geometrically similar conformations, taking both RMSD and energy difference into account in the kernel can better describe conformational transitions between neighboring conformations and lead to accurate embedding. We applied our diffusion map method to the ß-hairpin of the B1 domain of streptococcal protein G and to Trp-cage. Our results in ß-hairpin show that the diffusion map embedding achieves better results with the hybrid kernel than that with the RMSD-based kernel in terms of free energy landscape characterization and a new correlation measure between the cluster center Euclidean distances in the reduced-dimension space and the reciprocals of the total net flow between these clusters. In addition, our diffusion map analysis of the ultralong molecular dynamics trajectory of Trp-cage has provided a unified view of its folding mechanism. These promising results demonstrate the effectiveness of our diffusion map approach in the analysis of the dynamics and thermodynamics of molecular systems. The hybrid geometry-energy criterion could be also useful as a general dynamic measure for other purposes.


Assuntos
Proteínas de Bactérias/química , Peptídeos/química , Algoritmos , Difusão , Cadeias de Markov , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Domínios Proteicos , Termodinâmica
12.
Biochimie ; 166: 52-76, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31505212

RESUMO

Trypsin and chymotrypsin-like serine proteases from family S1 (clan PA) constitute the largest protease group in humans and more generally in vertebrates. The prototypes chymotrypsin, trypsin and elastase represent simple digestive proteases in the gut, where they cleave nearly any protein. Multidomain trypsin-like proteases are key players in the tightly controlled blood coagulation and complement systems, as well as related proteases that are secreted from diverse immune cells. Some serine proteases are expressed in nearly all tissues and fluids of the human body, such as the human kallikreins and kallikrein-related peptidases with specialization for often unique substrates and accurate timing of activity. HtrA and membrane-anchored serine proteases fulfill important physiological tasks with emerging roles in cancer. The high diversity of all family members, which share the tandem ß-barrel architecture of the chymotrypsin-fold in the catalytic domain, is conferred by the large differences of eight surface loops, surrounding the active site. The length of these loops alters with insertions and deletions, resulting in remarkably different three-dimensional arrangements. In addition, metal binding sites for Na+, Ca2+ and Zn2+ serve as regulatory elements, as do N-glycosylation sites. Depending on the individual tasks of the protease, the surface loops determine substrate specificity, control the turnover and allow regulation of activation, activity and degradation by other proteins, which are often serine proteases themselves. Most intriguingly, in some serine proteases, the surface loops interact as allosteric network, partially tuned by protein co-factors. Knowledge of these subtle and complicated molecular motions may allow nowadays for new and specific pharmaceutical or medical approaches.


Assuntos
Serina Endopeptidases/química , Serina Endopeptidases/fisiologia , Animais , Sítios de Ligação , Domínio Catalítico , Humanos , Modelos Moleculares , Conformação Proteica em Folha beta , Relação Estrutura-Atividade , Especificidade por Substrato
13.
Molecules ; 24(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505731

RESUMO

To expand the utilization of oyster protein (OP), the effects of high pressure (100 to 500 MPa) on chemical forces, structure, microstructure, and digestibility properties were investigated. High pressure (HP) treatment enhanced the electrostatic repulsion (from -13.3Control to -27.8HP200 mV) between protein molecules and avoided or retarded the formation of protein aggregates. In addition, the HP treated samples showed uniform distribution and small particle size. The changes in electrostatic interaction and particle size contributed to the improvement of solubility (from 10.53%Control to 19.92%HP500 at pH 7). The stretching and unfolding of protein were modified by HP treatment, and some internal hydrophobic groups and -SH groups were exposed. HP treatment modified the secondary structure of OP. The treated samples contained less α-helix and ß-sheet structures, whereas the proportions of ß-sheet and random coil structures were increased. The treated samples have high digestibility in the stomach (from 26.3%Control to 39.5%HP500) and in the total digestive process (from 62.1%Control to 83.7%HP500). In addition, the total digestive production showed higher percentages of small peptides (<1 kDa) after HP treatment. The protein solubility and digestibility were increased after HP treatment, and high solubility and high digestibility might increase the chance that OP become a kind of protein supplement.


Assuntos
Ostreidae/química , Pressão/efeitos adversos , Agregados Proteicos , Proteínas/química , Animais , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Estrutura Secundária de Proteína , Proteínas/metabolismo , Solubilidade , Estômago/química
14.
Phys Chem Chem Phys ; 21(36): 20239-20251, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31490518

RESUMO

The amyloid formation of human islet amyloid polypeptide (hIAPP)-an intrinsically disordered peptide, is associated with type II diabetes. Cellular membranes, especially those composed of negatively-charged lipids, accelerate the hIAPP amyloid fibrillation, and their integrity is disrupted during the aggregation process, leading to cell apoptosis. However, the underlying molecular mechanism is not well understood. Herein, we investigated the conformational dynamics during the interactions of hIAPP monomer with POPG membrane bilayer, by carrying out µs-long all-atom molecular dynamics simulations. Starting from the metastable coiled conformations in water, hIAPP monomers tend to adopt transient α-helical and ß-sheet structures when adsorbing to the membrane surface. The amphiphilic N-terminal region further inserts into the membrane interior and is located at the lipid head-tail interface, mainly in turn and α-helical structures. In contrast, the ß-hairpin structures reside on the membrane surface without insertion, and expand laterally with the hydrophobic residues exposed to the solvent. Moreover, the adsorption and insertion of hIAPP monomers induce two distinct local membrane deformations: (1) the hIAPP adsorption on the membrane surface mainly causes membrane bending; (2) the insertion of both turns and α-helices synchronizes with the formation of hydrophobic defects on the POPG membrane, leading to stronger membrane stretching and a longer coherence length of membrane thinning. Based on the structural and dynamical results, we propose that ß-hairpin structures may be a precursor for the fibrillation on the membrane surface due to the flat geometry and hydrophobic regions exposed to solvent, while N-terminal amphiphilic α-helices would facilitate hIAPP assembling into toxic oligomers inside the membrane.


Assuntos
Membrana Celular/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo
15.
Molecules ; 24(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470504

RESUMO

Brucellosis, also known as "undulant fever" is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.


Assuntos
Proteínas de Bactérias/química , Brucella/química , DNA/química , Fatores de Virulência/química , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Brucella/patogenicidade , DNA/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Motivos de Nucleotídeos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Homologia Estrutural de Proteína , Termodinâmica , Fatores de Virulência/metabolismo
16.
DNA Cell Biol ; 38(10): 1056-1068, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31403329

RESUMO

The AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) transcription factor represents one of the largest plant-specific transcriptional regulators in plants. ERF plays important roles in the regulation of various developmental processes and acts as a mediator in plant external stress responses. However, the research of the ERF gene family is still limited in alfalfa (Medicago sativa L.), one of the most important forage legume species in the world. In the present study, a total of 159 ERF genes were identified, and the phylogenetic reconstruction, classification, conserved motifs, signal peptide prediction, and expression patterns under salt, drought, and low-temperature stresses of these ERF genes were comprehensively analyzed. The ERF genes family in alfalfa could be classified into 10 groups and predicted to be strongly homologous. Based on the structure and functions relationships, the III and IV subfamilies were more likely to play functions in abiotic stresses and 18 MsERF genes were selected for further quantitative real-time PCR validation in different stresses treatment. The results showed that all these MsERF genes were upregulated under three stresses except MsERF008. This study identified the possibility of abiotic tolerance candidate genes playing various roles in stress resistance at the whole-genome level, which would provide primary understanding for exploring ERF-mediated tolerance in alfalfa.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Medicago sativa/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Temperatura Baixa , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Secas , Perfilação da Expressão Gênica , Medicago sativa/classificação , Medicago sativa/metabolismo , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Mapeamento de Interação de Proteínas , Isoformas de Proteínas , Salinidade , Estresse Fisiológico
17.
Chem Commun (Camb) ; 55(70): 10392-10395, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31407730

RESUMO

The preference of N,N-aryl, alkyl tertiary amides for cis conformations has been exploited through the use of tertiary squaramides as hairpin turn units that promote the folding of aromatic ß-sheets. Head-to-head aromatic arrangements were shown to prevail in sufficiently long bent aromatic sequences.


Assuntos
Dobramento de Proteína , Proteínas/química , Quinina/análogos & derivados , Cristalografia por Raios X , Conformação Proteica em Folha beta , Quinina/química
18.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370143

RESUMO

Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% ß-strand structures, respectively. The structure of BmADK was stable in pH 5.0-11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.


Assuntos
Adenosina Quinase/genética , Bombyx/genética , Proteínas de Insetos/genética , Larva/genética , Pupa/genética , Adenosina/química , Adenosina/metabolismo , Adenosina Quinase/química , Adenosina Quinase/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bombyx/enzimologia , Bombyx/crescimento & desenvolvimento , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Cinética , Larva/enzimologia , Larva/crescimento & desenvolvimento , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pupa/enzimologia , Pupa/crescimento & desenvolvimento , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
19.
Nucleic Acids Res ; 47(16): 8888-8898, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372631

RESUMO

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.


Assuntos
Apoproteínas/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
20.
Nucleic Acids Res ; 47(16): 8337-8347, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372632

RESUMO

DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome.


Assuntos
Enzimas Reparadoras do DNA/química , Reparo do DNA , DNA Viral/química , Proteínas de Ligação a DNA/química , DNA/química , Dímeros de Pirimidina/química , Bacteriófago lambda/química , Bacteriófago lambda/genética , Sítios de Ligação , DNA/genética , DNA/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Difusão , Humanos , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Concentração Osmolar , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Dímeros de Pirimidina/metabolismo , Imagem Individual de Molécula
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