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1.
Biochimie ; 2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34634369

RESUMO

The transmembrane emp24 domain-containing (TMED) proteins, also called p24 proteins, are members of a family of sorting receptors present in all representatives of the Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMED's structure across different subfamilies. Besides, although the presence of a TMED homo-oligomerization was suggested previously based on crystallographic contacts observed for the isolated Golgi Dynamics (GOLD) domain, no further analyses of its presence in solution were done. Here, we describe the first high-resolution structure of a TMED1 GOLD representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers in solution even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane, are also presented.

2.
Biophys Chem ; 279: 106690, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34600312

RESUMO

GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor. When bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane-anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented. The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the protein's secondary structure, according to our circular dichroism data, it had a significant impact on the protein's thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.

3.
ACS Appl Mater Interfaces ; 13(2): 2289-2302, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33405500

RESUMO

Photodynamic therapy (PDT) applications are limited by the low penetration of UV-visible light into biological tissues. Considering X-rays as an alternative to excite photosensitizers (PS) in a deeper tumor, an intermediate particle able to convert the X-ray energy into visible light (scintillating nanoparticle, ScNP) is necessary. Moreover, accumulation of PS in the target cells is also required. Genetically encoded proteins could be used as a photosensitizer, allowing the exclusive expression of PS inside the tumor cells. Here, the interaction of eGFP, KillerOrange, and KillerRed proteins with LaF3:Tb3+ ScNP was investigated, for the first time, in terms of its physicochemical and energy transfer properties. The protein structure, stability, and function were evaluated upon adverse physiological conditions and X-ray irradiation. Optimal parameters for energy transfer from ScNP to the proteins were investigated, paving the way for the use of genetically encoded photosensitizers for applications in X-ray activated photodynamic therapy.


Assuntos
Fluoretos/química , Lantânio/química , Proteínas Luminescentes/química , Nanopartículas/química , Fármacos Fotossensibilizantes/química , Térbio/química , Linhagem Celular Tumoral , Transferência de Energia , Humanos , Proteínas Luminescentes/genética , Modelos Moleculares , Nanopartículas/ultraestrutura , Neoplasias/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/metabolismo , Raios X
4.
Int J Biol Macromol ; 164: 3632-3644, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32871120

RESUMO

Golgi Reassembly and Stacking Proteins (GRASPs) were firstly described as crucial elements in determining the structure of the Golgi complex. However, data have been accumulating over the years showing GRASPs can participate in various cell processes beyond the Golgi maintenance, including cell adhesion and migration, autophagy and unconventional secretion of proteins. A comprehensive understanding of the GRASP functions requires deep mechanistic knowledge of its structure and dynamics, especially because of the unique structural plasticity observed for many members of this family coupled with their high promiscuity in mediating protein-protein interactions. Here, we critically review data regarding the structural biophysics of GRASPs in the quest for understanding the structural determinants of different functionalities. We dissect GRASP structure starting with the full-length protein down to its separate domains (PDZ1, PDZ2 and SPR) and outline some structural features common to all members of the GRASP family (such as the presence of many intrinsically disordered regions). Although the impact of those exquisite properties in vivo will still require further studies, it is possible, from our review, to pinpoint factors that must be considered in future interpretation of data regarding GRASP functions, thus bringing somewhat new perspectives to the field.


Assuntos
Biofísica , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi/ultraestrutura , Conformação Proteica , Cristalografia por Raios X , Complexo de Golgi/química , Proteínas da Matriz do Complexo de Golgi/química , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura
5.
Int J Biol Macromol ; 162: 1982-1993, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32822731

RESUMO

Golgi Reassembly and Stacking Proteins (GRASPs), including GRASP65/GRASP55, were firstly found as stacking factors of Golgi cisternae. Their involvement in other processes, such as unconventional protein secretion (UPS), have been demonstrated, suggesting GRASPs act as interaction hubs. However, structural details governing GRASP functions are not understood thoroughly. Here, we explored the structural features of human cis-Golgi GRASP65 in aqueous solution and compared them with those from trans-Golgi GRASP55. Besides their distinct Golgi localization, GRASP65/55 also seem to be selectively recruited to mitosis-related events or to UPS. Despite preserving the monomeric form in solution seen for GRASP55, as inferred from our SEC-MALS and DLS data, GRASP65 exhibited higher intrinsic disorder and susceptibility to denaturant than GRASP55 (disorder prediction, urea denaturation and circular dichroism data). Moreover, spectroscopic and microscopic studies showed for GRASP65 the same temperature-dependent amorphous aggregation and time-dependent amyloid fibrillation at 37 °C seen for GRASP55. In the latter case, however, GRASP65 presented a lower aggregation rate than GRASP55. The present and previous data evidenced that intrinsic disorder and formation of higher-order oligomers, such as amyloid fibrils, are common features within GRASP family potentially impacting the protein's participation in cell processes.


Assuntos
Proteínas da Matriz do Complexo de Golgi/química , Proteínas Intrinsicamente Desordenadas/química , Proteínas de Membrana/química , Amiloide/metabolismo , Complexo de Golgi/metabolismo , Humanos , Transporte Proteico
6.
FEBS J ; 287(15): 3255-3272, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31920006

RESUMO

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix that includes the Golgi reassembly and stacking protein (GRASP), which was shown to be involved in cisternae stacking and lateral linkage in metazoan. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different binding partners. The conserved N terminus of the GRASP family includes two PSD-95, DLG, and ZO-1 (PDZ) domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content. However, PDZ1 alone mediates nearly all interactions between GRASPs and their partners. In this work, NMR, synchrotron radiation CD, and molecular dynamics (MD) were used to examine the structure, flexibility, and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual ß3 α1 ß4 ß5 α2 ß6 ß1 ß2 secondary structural arrangement and NMR data indicate that the PDZ1 binding pocket is formed by a stable ß2 -strand and a more flexible and unstable α2 -helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using MD simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.


Assuntos
Proteínas da Matriz do Complexo de Golgi/química , Proteínas da Matriz do Complexo de Golgi/metabolismo , Domínios PDZ , Conformação Proteica , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Homologia de Sequência
7.
J Inorg Biochem ; 203: 110930, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31812025

RESUMO

This work presents the synthesis and characterization of eight copper complexes [Cu(L-dipeptide)(neo)]·nH2O (neo = neocuproine) and their cytotoxic activities against tumor cell lines. The crystalline structure of [Cu(gly-val)(neo)]·3H2O, [Cu(gly-leu)(neo)]·H2O, [Cu(ala-gly)(neo)]·4H2O, [Cu(val-phe)(neo)]·4.5H2O and [Cu(phe-phe)(neo)]·3H2O were determined by single crystal X-ray diffraction. In all of them, the Cu(II) is pentacoordinated, in a square pyramidal environment. The coordination observed in solid state was retained in the major species in aqueous solution, as suggested by Electronic Paramagnet Resonance and UV-vis spectroscopies. The complexes were shown to have affinity for isolated DNA, as determined by Circular Dichroism experiments. Furthermore, biological experiments showed that all the complexes present high cytotoxic activity against the cell lines: MDA-MB-231, MCF-7 (human metastatic breast adenocarcinomas, the first triple negative), MCF-10A (human normal breast cells), A549 (human lung epithelial carcinoma) and MRC-5 (human lung epithelial cells). Together, these results suggest that these compounds are promising steps towards new effective drugs to treat cancer.


Assuntos
Antineoplásicos/síntese química , Quelantes/síntese química , Complexos de Coordenação/síntese química , Cobre/química , Dipeptídeos/química , Fenantrolinas/química , Células A549 , Antineoplásicos/toxicidade , Proliferação de Células/efeitos dos fármacos , Quelantes/toxicidade , Complexos de Coordenação/toxicidade , DNA/química , Humanos , Células MCF-7 , Neoplasias de Mama Triplo Negativas/metabolismo
8.
FEBS J ; 286(17): 3340-3358, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31044497

RESUMO

The Golgi complex is part of the endomembrane system and is responsible for receiving transport cargos from the endoplasmic reticulum and for sorting and targeting them to their final destination. To perform its function in higher eukaryotic cells, the Golgi needs to be correctly assembled as a flattened membrane sandwich kept together by a protein matrix. The precise mechanism controlling the Golgi cisternae assembly is not yet known, but it is widely accepted that the Golgi Reassembly and Stacking Protein (GRASP) is a main component of the Golgi protein matrix. Unlike mammalian cells, which have two GRASP genes, lower eukaryotes present only one gene and distinct Golgi cisternae assembly. In this study, we performed a set of biophysical studies to get insights on the structural properties of the GRASP domains (DGRASPs) from both human GRASP55 and GRASP65 and compare them with GRASP domains from lower eukaryotes (Saccharomyces cerevisiae and Cryptococcus neoformans). Our data suggest that both human DGRASPs are essentially different from each other and that DGRASP65 is more similar to the subgroup of DGRASPs from lower eukaryotes in terms of its biophysical properties. GRASP55 is present mainly in the Golgi medial and trans faces, which are absent in both fungi, while GRASP65 is located in the cis-Golgi. We suggest that the GRASP65 gene is more ancient and that its paralogue GRASP55 might have appeared later in evolution, together with the medial and trans Golgi faces in mammalians.


Assuntos
Proteínas Fúngicas/química , Proteínas da Matriz do Complexo de Golgi/química , Homologia Estrutural de Proteína , Cryptococcus neoformans , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas da Matriz do Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi/metabolismo , Saccharomyces cerevisiae
9.
Food Sci Nutr ; 7(4): 1353-1360, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31024708

RESUMO

The in vitro antioxidant properties of golden grass (GG), a grass-like herb (Syngonanthus nitens), were investigated by electron paramagnetic resonance (EPR) spectroscopy. We measured the antioxidant capacity of methanolic extracts based on their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The kinetics of reaction between DPPH and GG extract was determined. This kinetics followed a biexponential decay, and this behavior was attributed to different flavonoids acting together as antioxidants. Isoorientin and luteolin, which are two of the eight flavonoids found in GG extract, were used to investigate kinetics of reaction between DPPH and both the flavonoids acting separately and together. The antioxidant activity of GG extract was determined in terms of the vitamin C equivalent antioxidant capacity (VCEAC). Compared to other well-known plant-based antioxidants, such as pulp and peels of fruit and vegetables, S. nitens presented a high antioxidant capacity (VCEAC = 1,485 ± 198 mg/100 g), indicating that it should be regarded as a valuable source of antioxidants and also that it may bestow health benefits when consumed.

10.
PLoS One ; 13(8): e0202148, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30125294

RESUMO

Thermophilic fungi are a promising source of thermostable enzymes able to hydrolytically or oxidatively degrade plant cell wall components. Among these enzymes are lytic polysaccharide monooxygenases (LPMOs), enzymes capable of enhancing biomass hydrolysis through an oxidative mechanism. Myceliophthora thermophila (synonym Sporotrichum thermophile), an Ascomycete fungus, expresses and secretes over a dozen different LPMOs. In this study, we report the overexpression and biochemical study of a previously uncharacterized LPMO (MtLPMO9J) from M. thermophila M77 in Aspergillus nidulans. MtLPMO9J is a single-domain LPMO and has 63% sequence similarity with the catalytic domain of NcLPMO9C from Neurospora crassa. Biochemical characterization of MtLPMO9J revealed that it performs C4-oxidation and is active against cellulose, soluble cello-oligosaccharides and xyloglucan. Moreover, biophysical studies showed that MtLPMO9J is structurally stable at pH above 5 and at temperatures up to 50°C. Importantly, LC-MS analysis of the peptides after tryptic digestion of the recombinantly produced protein revealed not only the correct processing of the signal peptide and methylation of the N-terminal histidine, but also partial autoxidation of the catalytic center. This shows that redox conditions need to be controlled, not only during LPMO reactions but also during protein production, to protect LPMOs from oxidative damage.


Assuntos
Ascomicetos/enzimologia , Polissacarídeos Fúngicos/metabolismo , Oxigenases de Função Mista/metabolismo , Sequência de Aminoácidos , Ascomicetos/genética , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Polissacarídeos Fúngicos/química , Oxigenases de Função Mista/química , Oxigenases de Função Mista/classificação , Oxigenases de Função Mista/genética , Filogenia , Análise de Sequência de DNA , Relação Estrutura-Atividade , Especificidade por Substrato , Espectrometria de Massas em Tandem , Termodinâmica
11.
Biochim Biophys Acta Gen Subj ; 1862(4): 855-865, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29339081

RESUMO

BACKGROUND: Golgi Reassembly and Stacking Proteins (GRASPs) are widely spread among eukaryotic cells (except plants) and are considered as key components in both the stacking of the Golgi cisternae and its lateral connection. Furthermore, GRASPs were also proved essential in the unconventional secretion pathway of several proteins, even though the mechanism remains obscure. It was previously observed that the GRASP homologue in Cryptococcus neoformans has a molten globule-like behavior in solution. METHODS: We used circular dichroism, synchrotron radiation circular dichroism and steady-state as well as time-resolved fluorescence. RESULTS: We report the disorder-to-order transition propensities for a native molten globule-like protein in the presence of different mimetics of cell conditions. Changes in the dielectric constant (such as those experienced close to the membrane surface) seem to be the major factor in inducing multiple disorder-to-order transitions in GRASP, which shows very distinct behavior when in conditions that mimic the vicinity of the membrane surface as compared to those found when free in solution. Other folding factors such as molecular crowding, counter ions, pH and phosphorylation exhibit lower or no effect on GRASP secondary structure and/or stability. GENERAL SIGNIFICANCE: To the best of our knowledge, this is the first study focusing on understanding the disorder-to-order transitions of a molten globule structure without the need of any mild denaturing condition. A model is also introduced aiming at describing how the cell could manipulate the GRASP sensitivity to changes in the dielectric constant during different cell-cycle periods.


Assuntos
Proteínas Fúngicas/química , Proteínas de Membrana/química , Conformação Proteica , Dobramento de Proteína , Álcoois/química , Álcoois/metabolismo , Dicroísmo Circular , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Complexo de Golgi/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Membrana/metabolismo , Metais/química , Metais/metabolismo , Modelos Moleculares , Desnaturação Proteica , Estrutura Secundária de Proteína , Termodinâmica , Água/química , Água/metabolismo
12.
Biotechnol Biofuels ; 10: 269, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163671

RESUMO

Background: The Bacillus subtilis endo-ß-1,4-glucanase (BsCel5A) hydrolyzes ß-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds ß-1,4-linked glucan. Methods: Here we investigate the BsCel5A ß-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having ß-1,3-1,4 glucan affinity. Results: The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley ß-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with ß-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with ß-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the ß-1,3-1,4 glucan. Conclusions: These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates.

13.
J Phys Chem B ; 121(49): 11085-11095, 2017 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-29148803

RESUMO

Human dihydroorotate dehydrogenase (HsDHODH) enzyme has been studied as selective target for inhibitors to block the enzyme activity, intending to prevent proliferative diseases. The N-terminal microdomain seems to play an important role in the enzyme function. However, the molecular mechanism of action and dynamics of this region are not totally understood yet. This study analyzes the interaction and conformation in model membranes of HsDHODH microdomain using peptide analogues containing the paramagnetic amino acid TOAC at strategic positions. In buffer solution, the analogues presented a disordered conformation, but acquired a high content of α-helical structure in membrane mimetics, which was found to be lipid dependent. The microdomain peptide structure in micelles showed a very different peptide conformation when compared to the reported crystal structure, displaying a conformational flexibility of its helices, promoted by the connecting loop, which might be functionally relevant. Electron spin resonance in membrane compositions containing POPC, POPE, and cardiolipin showed that interaction of the analogues was enhanced by the presence of cardiolipin, indicating that the microdomain preferentially interacts with cardiolipin-containing membranes. Therefore, the great flexibility of the microdomain and the cardiolipin affinity should be considered in further studies aimed at finding new inhibitory compounds to fight proliferative diseases.


Assuntos
Lipídeos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Peptídeos/química , Humanos , Micelas , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Conformação Proteica
14.
Int J Biol Macromol ; 102: 42-51, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28359888

RESUMO

Nifurtimox and benznidazole represent the only treatments options available targeting Chagas disease, the most important parasitic infection in the Americas. However, use of these is problematic as they are toxic and ineffective against the more severe stages of the disease. In this work, we used a multidisciplinary approach to characterise the fumarases from Trypanosoma cruzi, the causative agent of Chagas Disease. We showed this trypanosome expresses cytosolic and mitochondrial fumarases that via an iron-sulfur cluster mediate the reversible conversion of fumarate to S-malate. Based on sequence, biochemical properties and co-factor binding, both T. cruzi proteins share characteristics with class I fumarases, enzymes found in bacteria and some other protozoa but absent from humans, that possess class II isoforms instead. Gene disruption suggested that although the cytosolic or mitochondrial fumarase activities are individually dispensable their combined activity is essential for parasite viability. Finally, based on the mechanistic differences with the human (host) fumarase, we designed and validated a selective inhibitor targeting the parasite enzyme. This study showed that T. cruzi fumarases should be exploited as targets for the development of new chemotherapeutic interventions against Chagas disease.


Assuntos
Fumarato Hidratase/metabolismo , Trypanosoma cruzi/enzimologia , Citosol/enzimologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Fumarato Hidratase/antagonistas & inibidores , Fumarato Hidratase/química , Mitocôndrias/enzimologia , Transporte Proteico , Trypanosoma cruzi/citologia
15.
Int J Biol Macromol ; 102: 284-296, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28390829

RESUMO

Acyl-CoA Binding Proteins (ACBP) form a housekeeping family of proteins that is responsible for the buffering of long chain acyl-coenzyme A esters (LCFA-CoA) inside the cell. Even though numerous studies have focused on the characterization of different members of the ACBP family, the knowledge about the impact of both LCFA-CoA and phospholipids on ACBP structure and stability remains scarce. Besides, there are still controversies regarding the possible interaction of ACBP with biological membranes, even though this might be essential for the cargo capture and delivery. In this study, we observed that LCFA-CoA and phospholipids play opposite roles on protein stability and that the interaction with the membrane is dictated by electrostatic interaction. Furthermore, the results support the hypothesis that the LCFA-CoA delivery is driven by the increase of the negative charge on the membrane surface. The combined influence played by the different molecules on ACBP structure is discussed on the light of cargo capture/delivery giving new insights about this important process.


Assuntos
Acil Coenzima A/química , Acil Coenzima A/farmacologia , Inibidor da Ligação a Diazepam/química , Inibidor da Ligação a Diazepam/metabolismo , Ésteres/química , Fosfolipídeos/química , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Inibidor da Ligação a Diazepam/genética , Mutação , Transição de Fase , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos
16.
Biochim Biophys Acta Biomembr ; 1859(6): 1133-1143, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28336314

RESUMO

Pulmonary surfactant exhibits phase coexistence over a wide range of surface pressure and temperature. Less is known about the effect of temperature on pulmonary surfactant models. Given the lack of studies on this issue, we used electron paramagnetic resonance (EPR) and nonlinear least-squares (NLLS) simulations to investigate the thermotropic phase behavior of the matrix that mimics the pulmonary surfactant lipid complex, i.e., the lipid mixture composed of dipalmitoyl phosphatidylcholine (DPPC), palmitoyl-oleoyl phosphatidylcholine (POPC) and palmitoyl-oleoyl phosphatidylglycerol (POPG). Irrespective of pH, the EPR spectra recorded from 5°C to 25°C in the DPPC/POPC/POPG (4:3:1) model membrane contain two spectral components corresponding to lipids in gel-like and fluid-like phases, indicating a coexistence of two domains in that range. The temperature dependence of the distribution of spin labels between the domains yielded nonlinear van't Hoff plots. The thermodynamic parameters evaluated were markedly different for DPPC and for the ternary DPPC/POPC/POPG (4:3:1) membranes and exhibited a dependence on chemical environment. While enthalpy and entropy changes for DPPC were always positive and presented a quadratic behavior with temperature, those of the ternary mixture were linearly dependent on temperature and changed from negative to positive values. Despite that, enthalpy-entropy compensation takes place in the two systems. The thermotropic process associated with the coexistence of the two domains is entropically-driven in DPPC and either entropically- or enthalpically-driven in the pulmonary surfactant membrane depending on the pH, ionic strength and temperature. The significance of these results to the structure and function of the pulmonary surfactant lipid matrix is discussed.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Modelos Químicos , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Surfactantes Pulmonares/química , Concentração de Íons de Hidrogênio , Membranas Artificiais , Concentração Osmolar , Transição de Fase , Propriedades de Superfície , Temperatura , Termodinâmica
17.
Sci Rep ; 6: 37131, 2016 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-27892522

RESUMO

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.


Assuntos
Membrana Celular/química , Fusão de Membrana , Vírus da SARS/química , Proteínas Virais de Fusão/química , Internalização do Vírus , Varredura Diferencial de Calorimetria , Espectroscopia de Ressonância de Spin Eletrônica , Peptídeos/química , Termodinâmica
18.
Sci Rep ; 6: 29976, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27436376

RESUMO

Among all proteins localized in the Golgi apparatus, a two-PDZ (PSD95/DlgA/Zo-1) domain protein plays an important role in the assembly of the cisternae. This Golgi Reassembly and Stacking Protein (GRASP) has puzzled researchers due to its large array of functions and relevance in Golgi functionality. We report here a biochemical and biophysical study of the GRASP55/65 homologue in Cryptococcus neoformans (CnGRASP). Bioinformatic analysis, static fluorescence and circular dichroism spectroscopies, calorimetry, small angle X-ray scattering, solution nuclear magnetic resonance, size exclusion chromatography and proteolysis assays were used to unravel structural features of the full-length CnGRASP. We detected the coexistence of regular secondary structures and large amounts of disordered regions. The overall structure is less compact than a regular globular protein and the high structural flexibility makes its hydrophobic core more accessible to solvent. Our results indicate an unusual behavior of CnGRASP in solution, closely resembling a class of intrinsically disordered proteins called molten globule proteins. To the best of our knowledge, this is the first structural characterization of a full-length GRASP and observation of a molten globule-like behavior in the GRASP family. The possible implications of this and how it could explain the multiple facets of this intriguing class of proteins are discussed.


Assuntos
Proteínas de Transporte/química , Proteínas de Membrana/química , Conformação Proteica , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Domínios PDZ , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Desdobramento de Proteína , Soluções , Relação Estrutura-Atividade
19.
Biophys Rev ; 8(2): 179-191, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28510056

RESUMO

Protein-membrane interactions play essential roles in a variety of cell functions such as signaling, membrane trafficking, and transport. Membrane-recruited cytosolic proteins that interact transiently and interfacially with lipid bilayers perform several of those functions. Experimental techniques capable of probing changes on the structural dynamics of this weak association are surprisingly limited. Among such techniques, electron spin resonance (ESR) has the enormous advantage of providing valuable local information from both membrane and protein perspectives by using intrinsic paramagnetic probes in metalloproteins or by attaching nitroxide spin labels to proteins and lipids. In this review, we discuss the power of ESR to unravel relevant structural and functional details of lipid-peripheral membrane protein interactions with special emphasis on local changes of specific regions of the protein and/or the lipids. First, we show how ESR can be used to investigate the direct interaction between a protein and a particular lipid, illustrating the case of lipid binding into a hydrophobic pocket of chlorocatechol 1,2-dioxygenase, a non-heme iron enzyme responsible for catabolism of aromatic compounds that are industrially released in the environment. In the second case, we show the effects of GPI-anchored tissue-nonspecific alkaline phosphatase, a protein that plays a crucial role in skeletal mineralization, and on the ordering and dynamics of lipid acyl chains. Then, switching to the protein perspective, we analyze the interaction with model membranes of the brain fatty acid binding protein, the major actor in the reversible binding and transport of hydrophobic ligands such as long-chain, saturated, or unsaturated fatty acids. Finally, we conclude by discussing how both lipid and protein views can be associated to address a common question regarding the molecular mechanism by which dihydroorotate dehydrogenase, an essential enzyme for the de novo synthesis of pyrimidine nucleotides, and how it fishes out membrane-embedded quinones to perform its function.

20.
Protein Expr Purif ; 118: 39-48, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26432949

RESUMO

Galectin-4 (Gal4), a tandem-repeat type galectin, is expressed in healthy epithelium of the gastrointestinal tract. Altered levels of Gal4 expression are associated with different types of cancer, suggesting its usage as a diagnostic marker as well as target for drug development. The functional data available for this class of proteins suggest that the wide spectrum of cellular activities reported for Gal4 relies on distinct glycan specificity and structural characteristics of its two carbohydrate recognition domains. In the present work, two independent constructs for recombinant expression of the C-terminal domain of human galectin-4 (hGal4-CRD2) were developed. His6-tagged and untagged recombinant proteins were overexpressed in Escherichia coli, and purified by affinity chromatography followed by gel filtration. Correct folding and activity of hGal4-CRD2 were assessed by circular dichroism and fluorescence spectroscopies, respectively. Diffraction quality crystals were obtained by vapor-diffusion sitting drop setup and the crystal structure of CRD2 was solved by molecular replacement techniques at 1.78 Å resolution. Our work describes the development of important experimental tools that will allow further studies in order to correlate structure and binding properties of hGal4-CRD2 and human galectin-4 functional activities.


Assuntos
Carboidratos/química , Galectina 4/química , Galectina 4/isolamento & purificação , Sítios de Ligação , Biofísica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Galectina 4/genética , Galectina 4/metabolismo , Expressão Gênica , Humanos , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
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