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1.
Cell Physiol Biochem ; 54(1): 1-14, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31916733

RESUMO

BACKGROUND/AIMS: Deubiquitinating enzymes (DUBs) are crucially involved in controlling signal transductions, and reverse ubiquitination by removing the ubiquitin from protein substrates. The Hippo signaling has an important role in tissue growth, cell proliferation, differentiation, and apoptosis. Since disruption of the Hippo signaling is associated with a number of diseases, it is imperative to investigate the molecular mechanism of the Hippo signaling. METHODS: DUB screening was performed using the kidney of the mouse unilateral ureteric obstruction (UUO) model to identify the cellular mechanism of the DUB-regulated Hippo signaling. In addition, kidney cells were used to confirm cell proliferation and protein levels in the Hippo signaling pathway. Densitometric analysis was conducted to compare the expression level of proteins using Image J. RESULTS: We found that YOD1, also known as OTU1, is downregulated in the mouse UUO model. We also demonstrated that YOD1 binds to and deubiquitinates neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4). Furthermore, we observed that YOD1 suppresses NEDD4-induced cell proliferation. CONCLUSION: YOD1 regulates the Hippo signaling pathway through NEDD4, and the K63-linked polyubiquitin chain of NEDD4 plays an important role. Also, our results indicate that YOD1 plays an important role in kidney diseases.


Assuntos
Ubiquitina-Proteína Ligases Nedd4/metabolismo , Transdução de Sinais , Tioléster Hidrolases/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Camundongos , Mutagênese , Ubiquitina-Proteína Ligases Nedd4/química , Ubiquitina-Proteína Ligases Nedd4/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Tioléster Hidrolases/química , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
2.
Food Chem ; 305: 125463, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520921

RESUMO

Protein conformation and the 3D water structure play important roles in the ability of bovine serum albumin (BSA) to form stable nanostructures with bioactive molecules. We studied the influence of BSA unfolding and those of two Hofmeister salts, sodium chloride (NaCl) as kosmotrope and sodium thiocyanate (NaSCN) as chaotrope, on BSA/lutein binding at pH 7.4 using fluorescence spectroscopy. The BSA/lutein complex formation was entropically driven and lutein was preferentially bound to site III of BSA. The binding constant (104 L mol-1), complex stoichiometry (1:1), and thermodynamic potential for BSA/lutein binding were independent of protein conformation and Hofmeister salts. However, the enthalpic and entropic components of BSA/lutein binding in the presence of NaSCN decreased as the temperature increased. The opposite was observed for BSA/lutein binding in the presence of NaCl and for denatured BSA/lutein binding. Therefore, the BSA conformation and 3D water structure directly affected the BSA/lutein binding thermodynamics.


Assuntos
Luteína/metabolismo , Sais/química , Soroalbumina Bovina/metabolismo , Animais , Sítios de Ligação , Bovinos , Luteína/química , Ligação Proteica , Conformação Proteica , Soroalbumina Bovina/química , Cloreto de Sódio/química , Espectrometria de Fluorescência , Temperatura Ambiente , Termodinâmica , Tiocianatos/química
3.
J Colloid Interface Sci ; 559: 1-12, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31605780

RESUMO

Experimental and computational approaches are utilized to investigate the influence of electrostatic fields on the binding force between human coagulation protein thrombin and its DNA aptamer. The thiolated aptamer was deposited onto gold substrate located in a liquid cell filled with binding buffer, then the thrombin-functionalized atomic force microscopy (AFM) probe was repeatedly brought into contact with the aptamer-coated surface under applied electrical potentials of -100, 0, and 100 mV respectively. Force drops during the pull-off process were measured to determine the unbinding forces between thrombin and aptamer in a range of loading rates spanning from ~3 × 102 to ~1 × 104 pN/s. The results from experiments showed that both of the binding strength and propensity of the complex are drastically diminished under positive electrode potential, whereas there is no influence on the molecular binding from negative electrode potential. We also used a theoretical analysis to explain the nature of electrostatic potential and field inside the aptamer-thrombin layer, which in turn could quantify the influence of the electrostatically repulsive force on a thrombin molecule that promotes dissociation from the aptamer due to positive electrode potential, and achieve good agreement with the experimental results. The study confirms the feasibility of electrostatic modulation upon the binding interaction between thrombin and aptamer, and implicates an underlying application perspective upon nanoscale manipulation of the stimuli responsive biointerface.


Assuntos
Aptâmeros de Nucleotídeos/química , Trombina/química , Técnicas Biossensoriais/métodos , Eletricidade , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Fenômenos Mecânicos , Modelos Moleculares , Ligação Proteica , Eletricidade Estática , Propriedades de Superfície
4.
Food Chem ; 307: 125514, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639576

RESUMO

The thermodynamics and kinetics of binding between human serum albumin (HSA) and resveratrol (RES) or its analog (RESAn1) were investigated by surface plasmon resonance (SPR). The binding constant and the kinetic constants of association and dissociation indicated that RESAn1 has higher affinity toward HSA than does RES. The formation of these complexes was entropically driven ( [Formula: see text] , [Formula: see text]  KJ mol-1). However, for both polyphenols, the activation energy (Eact) of association (a) of free molecules was higher than that for dissociation (d) of the stable complex ( [Formula: see text]  KJ mol-1), and the rate of association was faster than that of dissociation since the activation Gibbs free energy (ΔG‡) was lower for the former (ΔGaHSA-RES‡â‰…54.73,ΔGdHSA-RES‡â‰…73.83,ΔGaHSA-RESAn1‡â‰…54.14,ΔGdHSA-RESAn1‡â‰…73.97 KJ mol-1). This study showed that small differences in the structure of polyphenols such as RES and RESAn1 influenced the thermodynamics and kinetics of the complex formation with HSA.


Assuntos
Fenóis/química , Resveratrol/metabolismo , Albumina Sérica Humana/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Ligação Proteica , Resveratrol/química , Albumina Sérica Humana/química , Ressonância de Plasmônio de Superfície , Temperatura Ambiente , Termodinâmica
5.
Biophys Chem ; 256: 106281, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31756663

RESUMO

Timely and accurate diagnosis of Alzheimer's disease (AD) remains a major challenge in the medical arena. ß-amyloid (Aß) imaging techniques such as positron emission tomography and single photon emission computed tomography require the use of an imaging probe. To date, only flutemetamol, florbetaben and florbetapir have been approved for clinical use as imaging probes. Design of imaging probes requires a detailed understanding of disease mechanism(s) and receptor-ligand interaction. In this study, molecular docking, molecular dynamics and binding free energies were used to investigate the multiple binding sites exhibited by ß-amyloid fibrils. Protein atomic models 2BEG, 5KK3, 2M4J, 2LMN, 5OQV, 2NAO, 2MVX and 2MXU (protein databank codes) were used to investigate the nature and location of binding sites and binding profiles of selected molecules with known affinities. Although amyloid fibrils are known to have multiple binding sites, we demonstrated that model 2MXU possesses one site which is druggable and can bind with common scaffolds currently being used in the imaging of amyloid fibrils. Models 2NAO, 5KK3 and 2M4J revealed that even though multiple sites may be available in some fibrils, the entire protein may not have a druggable site. Molecular dynamics revealed atomic models 2MXU and 2MVX to be the least flexible among the list. The outcomes of this investigation can be translated to assist in designing novel molecules that can be used for brain imaging in Alzheimer's disease.


Assuntos
Amiloide/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/metabolismo , Sítios de Ligação , Bases de Dados de Proteínas , Humanos , Ligantes , Ligação Proteica , Estrutura Terciária de Proteína
6.
Chem Commun (Camb) ; 55(92): 13864-13867, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31670736

RESUMO

The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.


Assuntos
Corynebacterium glutamicum/metabolismo , Heme/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Heme/química , Ligação Proteica , Estrutura Terciária de Proteína
7.
J Chem Phys ; 151(19): 195102, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31757139

RESUMO

Here, we investigate the unfolding behavior of a streptomycin-binding ribonucleic acid (RNA) aptamer under application of force in shear geometry. Using Langevin out-of-equilibrium simulations to emulate the single-molecule force spectroscopy (SMFS) experiment, we were able to understand the hierarchical unfolding process that occurs in the RNA molecule under application of stretching force and the influence of streptomycin modifying this unfolding. Subsequently, the application of the Jarzynski equality to the force profiles obtained in the pulling simulations shows that the free energies for individual systems and the difference of unfolding free energy upon streptomycin binding to the RNA free aptamer are in fair agreement with the experimental values, obtained through SMFS by Nick et al. [J. Phys. Chem. B 120, 6479 (2016)].


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Estreptomicina/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Termodinâmica
8.
Curr Top Med Chem ; 19(21): 1872-1876, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31696799

RESUMO

PPIs are involved in diverse biochemical events and perform their functions through the formation of protein-protein complexes or PPI networks. The large and flat interacting surfaces of PPIs make discovery of small-molecule modulators a challenging task. New strategies and more effective chemical technologies are needed to facilitate the development of PPIs small-molecule inhibitors. Covalent modification of a nucleophilic residue located proximally to the immediate vicinity of PPIs can overcome the disadvantages of large interacting surfaces and provides high-affinity inhibitors with increased duration of action and prolonged target modulation. On the other hand, covalent inhibitors that target non-conserved protein residues demonstrate improved selectivity over related protein family members. Herein, we highlight the latest progress of small-molecule covalent PPIs inhibitors and hope to shed light on future PPIs inhibitor design and development. The relevant challenges and opportunities are also discussed.


Assuntos
Proteínas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química
9.
Chem Commun (Camb) ; 55(93): 14074-14077, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31696869

RESUMO

We discovered that the function of cytochrome C can be modulated by DNA nanoribbons. Meanwhile, the interplay between the DNA nanoribbons and the native cytochrome C and the possible mechanisms are also discussed.


Assuntos
Citocromos c/metabolismo , DNA/metabolismo , Nanoestruturas/química , Peroxidases/metabolismo , Animais , Catálise , Citocromos c/química , DNA/química , Guaiacol/química , Cavalos , Oxirredução , Peroxidases/química , Ligação Proteica
10.
Adv Exp Med Biol ; 1163: 45-64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31707699

RESUMO

This chapter focuses on protein kinases that transfer the phosphate group of ATP to the hydroxyl group of a substrate protein. Five hundred eighteen human protein kinases are classified into serine/threonine kinases and tyrosine kinases and individually or synergistically transduce physiologic stimuli into cell to promote cell proliferation or apoptosis, etc. Protein kinases are identified as drug targets because dysfunction of kinases leads to severe diseases such as cancers and autoimmune diseases. A large number of the crystal structures of the protein kinase inhibitor complex are available in Protein Data Bank and facilitated the drug discovery targeting protein kinases. The protein kinase inhibitors are classified into categories, Type-I, Type-II, Type-III, Type-IV, and Type-V, and as a separate class, covalent-type inhibitors. In any type, a protein kinase inhibitor bound to the allosteric region is advantageous in terms of selectivity compared to the traditional ATP-competitive one. In the following sections, the successful and promising examples of the partially or fully allosteric protein kinase inhibitors are illustrated in the following pages.


Assuntos
Desenho de Drogas , Inibidores de Proteínas Quinases , Proteínas Quinases , Trifosfato de Adenosina , Descoberta de Drogas , Ativação Enzimática/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo
11.
Adv Exp Med Biol ; 1163: 279-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31707708

RESUMO

Allostery is a basic principle that enables proteins to process and transmit cellular information. Protein kinases evolved allosteric mechanisms to transduce cellular signals to downstream signalling components or effector molecules. Protein kinases catalyse the transfer of the terminal phosphate from ATP to protein substrates upon specific stimuli. Protein kinases are targets for the development of small molecule inhibitors for the treatment of human diseases. Drug development has focussed on ATP-binding site, while there is increase interest in the development of drugs targeting alternative sites, i.e. allosteric sites. Here, we review the mechanism of regulation of protein kinases, which often involve the allosteric modulation of the ATP-binding site, enhancing or inhibiting activity. We exemplify the molecular mechanism of allostery in protein kinases downstream of PI3-kinase signalling with a focus on phosphoinositide-dependent protein kinase 1 (PDK1), a model kinase where small compounds can allosterically modulate the conformation of the kinase bidirectionally.


Assuntos
Desenvolvimento de Medicamentos , Fosfatidilinositol 3-Quinases , Proteínas Quinases , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Adv Exp Med Biol ; 1163: 313-334, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31707709

RESUMO

Protein-protein interactions (PPIs) represent promising drug targets of broad-spectrum therapeutic interests due to their critical implications in both health and disease circumstances. Hence, they are widely accepted as the Holy Grail of drug development. Historically, PPIs were rendered "undruggable" for their large, flat, and pocket-less structures. Current attempts to drug these "intractable" targets include orthosteric and allosteric methodologies. Previous efforts employing orthosteric approaches like protein therapeutics and orthosteric small molecules frequently suffered from poor performance caused by the difficulties in directly targeting PPI interfaces. As structural biology progresses rapidly, allosteric modulators, which direct to the allosteric regulatory sites remote to the PPI surfaces, have gradually established as a potential solution. Allosteric pockets are topologically distal from the PPI orthosteric sites, and their ligands do not need to compete with the PPI partners, which helps to improve the physiochemical and pharmacological properties of allosteric PPI modulators. Thus, exploiting allostery to tailor PPIs is regarded as a tempting strategy in future PPI drug discovery. Here, we provide a comprehensive review of our representative achievements along the way we utilize allosteric effects to tame the difficult PPI systems into druggable targets. Importantly, we provide an in-depth mechanistic analysis of this success, which will be instructive to future related lead optimizations and drug design. Finally, we discuss the current challenges in allosteric PPI drug discovery. Their solutions as well as future perspectives are also presented.


Assuntos
Sítio Alostérico , Descoberta de Drogas , Regulação Alostérica , Sítio Alostérico/fisiologia , Ligantes , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia
13.
Adv Exp Med Biol ; 1163: 335-357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31707710

RESUMO

The allosteric property of globular proteins is applauded as their intrinsic ability to regulate distant sites, and this property further plays a critical role in a wide variety of cellular regulatory mechanisms. Recent advancements and studies have revealed the manifestation of allostery in intrinsically disordered proteins or regions as allosteric sites present within or mediated by IDP/IDRs facilitates the signaling interactions for various biological mechanisms which would otherwise be impossible for globular proteins to regulate. This thematic review has highlighted the biological outcomes that can be achieved by the mechanism of allosteric regulation of intrinsically disordered proteins or regions. The similar mechanism has been implemented on Adenovirus 5 early region 1A and tumor apoptosis protein p53 in correspondence with other partners in binary and ternary complexes, which are the subject of the current review. Both these proteins regulate once they bind to their partners, consequently, forming either a binary or a ternary complex. Allosteric regulation by IDPs is currently a subject undergoing intense study, and the ongoing research work will ensure a better understanding of precision and efficiency of cellular regulation by them. Allosteric regulation mechanism can also be researched by intrinsically disordered protein-specific force field.


Assuntos
Proteínas Intrinsicamente Desordenadas , Regulação Alostérica , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Transdução de Sinais
15.
Protein Pept Lett ; 26(10): 751-757, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618170

RESUMO

BACKGROUND: NMAAP1 plays a role in regulating macrophage differentiation to the M1 type and exerting antitumoral functions. It is not clear what role and mechanism NMAAP1 does play in the reversal of macrophages from M1 to M2. METHODS: We detected the typing of macrophages with high or low expression of NMAAP1 by QPCR and ELISA, and detected the colocalization of NMAAP1 and endogenous IP3R by laser confocal microscopy, and detected the protein expression in cells by Western-blotting. RESULTS: Our study found that knockdown NMAAP1 in RAW264.7 cells induced macrophage polarization to the M2 type and up-regulation of NMAAP1 in RAW264.7 cells maintain M1 Phenotype even in the presence of IL-4, a stronger inducer of the M2 type. Additionally, Coimmunoprecipitation revealed a protein-protein interaction between NMAAP1 and IP3R and then activates key molecules in the PKC-dependent Raf/MEK/ERK and Ca2+/CaM/CaMKII signaling pathways. Activation of PKC (Thr638/641), ERK1/2 (Thr202/Tyr204) and CaMKII (Thr286) is involved in the regulation of cell differentiation. CONCLUSION: NMAAP1 interacts with IP3R, which in turn activates the PKC-dependent Raf/MEK/ERK and Ca2+/CaM/CaMKII signaling pathways. These results provide a new explanation of the mechanism underlying M1 differentiation.


Assuntos
Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Macrófagos/citologia , Proteínas de Membrana/genética , Camundongos , Fenótipo , Ligação Proteica , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Regulação para Cima
16.
Protein Pept Lett ; 26(10): 768-775, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618171

RESUMO

INTRODUCTION: Metallothioneins (MTs) are members of a family of low molecular weight and cysteine-rich proteins that are involved in heavy metal homeostasis and detoxification in living organisms. Plants have multiple MT types that are generally divided into four subgroups according to the arrangement of Cys residues. METHODS: In the present study the E. coli cells which heterologously express four different rice MT (OsMT) isoforms were analyzed for the accumulation of two forms of chromium, Cr3+ and Cr6+. RESULTS: The results show that the transgenic bacteria were more tolerant than control cells when they were grown up in the medium comprising Cr(NO3)3.9H2O or Na2CrO4. The cells expressing OsMT1, OsMT2, OsMT3 and OsMT4 give rise to 6.5-, 2.7-, 5.5- and 2.1-fold improvements on the accumulation capacity for Cr3+ and 9-, 3-, 5- and 3- fold Cr6+ respectively compared with comparison to the control strain. Furthermore, the purified recombinant GST-OsMTs were tested for their binding ability to Cr+3 and Cr+6 in vitro. DISCUSSION: The data show that the recombinant GST-OsMT1 and GST-OsMT2 were able to bind both Cr3+ and Cr6+, in vitro. However, their binding strength was low with respect to previous tested divalent ions like Cd2+.


Assuntos
Cromo/química , Metalotioneína/química , Oryza/química , Proteínas de Plantas/química , Proteínas Recombinantes/química , Sequência de Aminoácidos , Cátions/química , Cisteína/química , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Metalotioneína/genética , Proteínas de Plantas/genética , Ligação Proteica , Isoformas de Proteínas , Proteínas Recombinantes/genética
17.
Protein Pept Lett ; 26(10): 792-797, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618172

RESUMO

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/ß- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.


Assuntos
Proteína Axina/química , Proteína Axina/genética , Proteínas Desgrenhadas/química , Domínios Proteicos/genética , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X/métodos , Proteínas Desgrenhadas/genética , Escherichia coli , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Via de Sinalização Wnt
18.
Plant Mol Biol ; 101(4-5): 499-506, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31621004

RESUMO

A simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different auxin response factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Compostos Orgânicos , Proteínas de Plantas/metabolismo , Soja/metabolismo , Fatores de Transcrição/metabolismo , Fluorescência , Ligação Proteica , Sensibilidade e Especificidade
19.
Photochem Photobiol Sci ; 18(11): 2657-2660, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31624823

RESUMO

Mr4511 from Methylobacterium radiotolerans is a 164 amino acid protein built of a flavin mononucleotide (FMN) binding, blue-light responsive LOV (Light, Oxygen, Voltage) core domain plus flanking regions. In contrast to the majority of LOV domains, Mr4511 lacks a tryptophan residue that was previously identified as a major quencher for the FMN triplet state in photosensitizers for singlet oxygen (SO) engineered from these photoreceptors. Here we show that for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitizer: both C71S and C71G variants exhibit SO quantum yields of formation, ΦΔ, around 0.2 in air-saturated solutions. Under oxygen saturated conditions, ΦΔ reaches ∼0.5 in deuterated buffer. The introduction of Trp112 in the canonical position for LOV domains dramatically lowers ΦΔ to values comparable to miniSOG, one of the early FMN binding proteins touted as a SO sensitizer. Besides its SO properties, Mr4511 is also exceedingly robust against denaturation with urea and is more photostable than free FMN.


Assuntos
Proteínas de Bactérias/metabolismo , Methylobacterium/metabolismo , Oxigênio Singlete/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Polarização de Fluorescência , Mutagênese Sítio-Dirigida , Oxigênio/química , Ligação Proteica , Teoria Quântica , Alinhamento de Sequência , Ureia/química
20.
J Chem Theory Comput ; 15(11): 5829-5844, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31593627

RESUMO

A powerful computational strategy to determine the equilibrium association constant of two macromolecules with explicit-solvent molecular dynamics (MD) simulations is the "geometric route", which considers the reversible physical separation of the bound complex in solution. Nonetheless, multiple challenges remain to render this type of methodology reliable and computationally efficient in practice. In particular, in one, formulation of the geometric route relies on the potential of mean force (PMF) for physically separating the two binding partners restrained along a straight axis, which must be selected prior to the calculation. However, practical applications indicate that the calculation of the separation PMF along the predefined rectilinear pathway may be suboptimal and slowly convergent. Recognizing that a rectilinear straight separation pathway is generally not representative of how the protein complex physically separates in solution, we put forth a novel theoretical framework for binding free-energy calculations, leaning on the optimal curvilinear minimum free-energy path (MFEP) determined from the string method. The proposed formalism is validated by comparing the results obtained using both rectilinear and curvilinear pathways for a prototypical host-guest complex formed by cucurbit[7]uril (CB[7]) binding benzene, and for the barnase-barstar protein complex. On the basis of multi-microsecond MD calculations, we find that the calculations following the traditional rectilinear pathway and the string-based curvilinear pathway agree quantitatively, but convergence is faster with the latter.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzeno/química , Benzeno/metabolismo , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Ligação Proteica , Proteínas/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , Termodinâmica
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