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1.
Comput Struct Biotechnol J ; 23: 473-482, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38261868

RESUMO

TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in Pichia pastoris using a novel medium-throughput Ca2+ influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target.

2.
J Extracell Vesicles ; 12(12): e12384, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-38031976

RESUMO

Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.


Assuntos
Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Leucócitos Mononucleares , Succinimidas/metabolismo , Linhagem Celular
3.
J Phys Chem Lett ; 14(14): 3368-3375, 2023 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-36995079

RESUMO

Genetic code expansion allows modification of the physical and chemical properties of proteins by the site-directed insertion of noncanonical amino acids. Here we exploit this technology for measuring nanometer-scale distances in proteins. (2,2'-Bipyridin-5-yl)alanine was incorporated into the green fluorescent protein (GFP) and used as an anchoring point for Cu(II) to create a spin-label. The incorporation of (2,2'-bipyridin-5-yl)alanine directly into the protein resulted in a high-affinity binding site for Cu(II) capable of outcompeting other binding positions in the protein. The resulting Cu(II)-spin label is very compact and not larger than a conventional amino acid. By using 94 GHz electron paramagnetic resonance (EPR) pulse dipolar spectroscopy we have been able to determine accurately the distance between two such spin-labels. Our measurements revealed that GFP dimers can adopt different quaternary conformations. The combination of spin-labeling using a paramagnetic nonconventional amino acid with high-frequency EPR techniques resulted in a sensitive method for studying the structures of proteins.


Assuntos
Alanina , Aminoácidos , Aminoácidos/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas de Fluorescência Verde , Marcadores de Spin , Cobre/química
4.
Membranes (Basel) ; 12(7)2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35877905

RESUMO

ATP2, a putative type 4 P-type ATPase, is a phosphatidylinositol-4-phosphate (PI4P)-regulated phospholipid transporter with an interesting potential as an antimalarial drug target due to its conservation across Plasmodium species and its essential role in the life cycle of Plasmodium falciparum. Despite its importance, the exact mechanism of its action and regulation is still not fully understood. In this study we used coarse-grained molecular dynamics (CG-MD) to elucidate the lipid-protein interactions between a heterogeneous lipid membrane containing phosphatidylinositol and Plasmodium chabaudi ATP2 (PcATP2), an ortholog of P. falciparum ATP2. Our study reveals structural information of the lipid fingerprint of ATP2, and provides structural information on the potential phosphatidylinositol allosteric binding site. Moreover, we identified a set of evolutionary conserved residues that may play a key role in the binding and stabilization of lipids in the binding pocket.

5.
Emerg Microbes Infect ; 10(1): 132-147, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33372863

RESUMO

Gene targeting approaches have demonstrated the essential role for the malaria parasite of membrane transport proteins involved in lipid transport and in the maintenance of membrane lipid asymmetry, representing emerging oportunites for therapeutical intervention. This is the case of ATP2, a Plasmodium-encoded 4 P-type ATPase (P4-ATPase or lipid flippase), whose activity is completely irreplaceable during the asexual stages of the parasite. Moreover, a recent chemogenomic study has situated ATP2 as the possible target of two antimalarial drug candidates. In eukaryotes, P4-ATPases assure the asymmetric phospholipid distribution in membranes by translocating phospholipids from the outer to the inner leaflet. In this work, we have used a recombinantly-produced P. chabaudi ATP2 (PcATP2), to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 associates with two of the three Plasmodium-encoded Cdc50 proteins: PcCdc50B and PcCdc50A. Purified PcATP2/PcCdc50B complex displays ATPase activity in the presence of either phosphatidylserine or phosphatidylethanolamine. In addition, this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work describes the first biochemical characterization of a Plasmodium lipid flippase, a first step towards the understanding of the essential physiological role of this transporter and towards its validation as a potential antimalarial drug target.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Membrana/metabolismo , Plasmodium/enzimologia , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Transporte Biológico , Clonagem Molecular , Hidrólise , Modelos Moleculares , Fosfolipídeos/metabolismo , Plasmodium/genética , Ligação Proteica , Conformação Proteica , ATPases Translocadoras de Prótons/química , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transformação Genética
6.
Int J Mol Sci ; 20(3)2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30764505

RESUMO

Constitutive or regulated membrane protein trafficking is a key cell biology process. Transient receptor potential channels are somatosensory proteins in charge of detecting several physical and chemical stimuli, thus requiring fine vesicular trafficking. The membrane proximal or pre-S1 domain (MPD) is a highly conserved domain in transient receptor potential channels from the vanilloid (TRPV) subfamily. MPD shows traits corresponding to protein-protein and lipid-protein interactions, and protein regulatory regions. We have expressed MPD of TRPV1 and TRPV2 as green fluorescente protein (GFP)-fusion proteins to perform an in vitro biochemical and biophysical characterization. Pull-down experiments indicate that MPD recognizes and binds Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptors (SNARE). Synchrotron radiation scattering experiments show that this domain does not self-oligomerize. MPD interacts with phosphatidic acid (PA), a metabolite of the phospholipase D (PLD) pathway, in a specific manner as shown by lipid strips and Trp fluorescence quenching experiments. We show for the first time, to the best of our knowledge, the binding to PA of an N-terminus domain in TRPV channels. The presence of a PA binding domain in TRPV channels argues for putative PLD regulation. Findings in this study open new perspectives to understand the regulated and constitutive trafficking of TRPV channels exerted by protein-protein and lipid-protein interactions.


Assuntos
Ácidos Fosfatídicos/metabolismo , Mapas de Interação de Proteínas , Canais de Cátion TRPV/metabolismo , Animais , Exocitose , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Proteínas SNARE/metabolismo , Canais de Cátion TRPV/química
7.
Curr Protoc Protein Sci ; 93(1): e59, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30021058

RESUMO

Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions. Consequently, the experimenter must pay attention to both the protein and the behavior of the detergent. This article provides a convenient protocol for estimating the CMC of detergents in given experimental conditions. Then, it presents two protocols aimed at monitoring the function of a membrane protein in the presence of detergent. Such experiments may help to test various detergents for their inactivating or stabilizing effects on long incubation times, ranging from few hours to some days. © 2018 by John Wiley & Sons, Inc.


Assuntos
Detergentes/química , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Micelas , Animais , Humanos , Estabilidade Proteica
8.
Methods Mol Biol ; 1586: 181-195, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28470605

RESUMO

Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in detergent micelles of the library's members.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Engenharia de Proteínas/métodos , Sistemas de Transporte de Aminoácidos/química , Bacillus subtilis/química , Proteínas de Bactérias/química , Detergentes/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Modelos Moleculares , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
9.
J Biol Chem ; 292(19): 7954-7970, 2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28302728

RESUMO

P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate. Using thrombin as the protease, Cdc50p remains intact and in complex with Drs2p, which is cleaved at two positions, namely after Arg104 and after Arg 1290, resulting in a homogeneous sample lacking 104 and 65 residues from its N and C termini, respectively. Removal of the 1291-1302-amino acid region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, whereas the 1-104 N-terminal residues have an additional but more modest significance for activity. The present results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can greatly increase its ATPase activity in the presence of PI4P and demonstrate that relief of such auto-inhibition remains compatible with subsequent regulation by PI4P. These experiments suggest that activation of the Drs2p-Cdc50p flippase follows a multistep mechanism, with preliminary release of a number of constraints, possibly through the binding of regulatory proteins in the trans-Golgi network, followed by full activation by PI4P.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Fosfatos de Fosfatidilinositol/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Arginina/química , Hidrólise , Mutação , Proteínas de Transferência de Fosfolipídeos/química , Fosfolipídeos/química , Fosforilação , Ligação Proteica , Domínios Proteicos , Proteólise , Trombina/química
10.
PLoS One ; 12(1): e0170481, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28118404

RESUMO

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.


Assuntos
Detergentes/farmacologia , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Micelas , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Difusão , Fluorometria , Glucosídeos/farmacologia , Halogenação , Cinética , Proteínas de Membrana/efeitos dos fármacos , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Solubilidade , Temperatura
11.
J Gen Physiol ; 147(4): 353-68, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26976827

RESUMO

The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis L-serine/L-threonine exchanger is the best-known prokaryotic paradigm of the mammalian L-amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.


Assuntos
Substituição de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Proteínas de Bactérias/química , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Proteica
12.
Biochem Biophys Res Commun ; 462(3): 221-6, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-25956061

RESUMO

Transient Receptor Potential (TRP) channels are related to adaptation to the environment and somatosensation. The transient receptor potential vanilloid (TRPV) subfamily includes six closely evolutionary related ion channels sharing the same domain organization and tetrameric arrangement in the membrane. In this study we have characterized biochemically TRPV2 channel membrane protein folding and transmembrane (TM) architecture. Deleting the first N-terminal 74 residues preceding the ankyrin repeat domain (ARD) show a key role for this region in targeting the protein to the membrane. We have demonstrated the co-translational insertion of the membrane-embedded region of the TRPV2 and its disposition in biological membranes, identifying that TM1-TM4 and TM5-TM6 regions can assemble as independent folding domains. The ARD is not required for TM domain insertion in the membrane. The folding features observed for TRPV2 may be conserved and shared among other TRP channels outside the TRPV subfamily.


Assuntos
Canais de Cátion TRPV/química , Animais , Repetição de Anquirina , Membrana Celular/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
13.
J Phys Chem B ; 116(48): 14023-8, 2012 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-23137163

RESUMO

In this work we have investigated the selectivity of lactose permease (LacY) of Escherichia coli (E. coli) for its surrounding phospholipids when reconstituted in binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). Förster resonance energy transfer (FRET) measurements have been performed to investigate the selectivity between a single tryptophan mutant of LacY used as donor (D), and two analogues of POPE and POPG labeled with pyrene in the acyl chains (Pyr-PE and Pyr-PG) used as acceptors. As a difference from previous works, now the donor has been single-W151/C154G/D68C LacY. It has been reported that the replacement of the aspartic acid in position 68 by cysteine inhibits active transport in LacY. The objectives of this work were to elucidate the phospholipid composition of the annular region of this mutant and to determine whether the mutation performed, D68C, induced changes in the protein-lipid selectivity. FRET efficiencies for Pyr-PE were always higher than for Pyr-PG. The values of the probability of each site in the annular ring being occupied by a label (µ) were similar at the studied temperatures (24 °C and 37 °C), suggesting that the lipid environment is not significantly affected when increasing the temperature. By comparing the results with those obtained for single-W151/C154G LacY, we observe that the mutation in the 68 residue indeed changes the selectivity of the protein for the phospholipids. This might be probably due to a change in the conformational dynamics of LacY.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Simportadores/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/genética , Fosfatidilgliceróis/metabolismo , Mutação Puntual , Simportadores/genética
14.
Proc Natl Acad Sci U S A ; 109(42): 16835-40, 2012 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-23033496

RESUMO

WT lactose permease of Escherichia coli (LacY) reconstituted into proteoliposomes loaded with a pH-sensitive fluorophore exhibits robust uphill H(+) translocation coupled with downhill lactose transport. However, galactoside binding by mutants defective in lactose-induced H(+) translocation is not accompanied by release of an H(+) on the interior of the proteoliposomes. Because the pK(a) value for galactoside binding is ∼10.5, protonation of LacY likely precedes sugar binding at physiological pH. Consistently, purified WT LacY, as well as the mutants, binds substrate at pH 7.5-8.5 in detergent, but no change in ambient pH is observed, demonstrating directly that LacY already is protonated when sugar binds. However, a kinetic isotope effect (KIE) on the rate of binding is observed, indicating that deuterium substitution for protium affects an H(+) transfer reaction within LacY that is associated with sugar binding. At neutral pH or pD, both the rate of sugar dissociation (k(off)) and the forward rate (k(on)) are slower in D(2)O than in H(2)O (KIE is ∼2), and, as a result, no change in affinity (K(d)) is observed. Alkaline conditions enhance the effect of D(2)O on k(off), the KIE increases to 3.6-4.0, and affinity for sugar increases compared with H(2)O. In contrast, LacY mutants that exhibit pH-independent high-affinity binding up to pH 11.0 (e.g., Glu325 → Gln) exhibit the same KIE (1.5-1.8) at neutral or alkaline pH (pD). Proton inventory studies exhibit a linear relationship between k(off) and D(2)O concentration at neutral and alkaline pH, indicating that internal transfer of a single H(+) is involved in the KIE.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Galactosidases/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Prótons , Simportadores/metabolismo , Deutério , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Galactosidases/química , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Mutação/genética , Ligação Proteica , Simportadores/química , Simportadores/genética
15.
Anal Biochem ; 423(1): 7-14, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22285978

RESUMO

Membrane proteins are challenging targets for structural biologists. Finding optimal candidates for such studies requires extensive and laborious screening of protein expression and/or stability in detergent. The use of green fluorescent protein (GFP) as a reporter has enormously facilitated these studies; however, its 238 residues can potentially alter the intrinsic properties of the target (e.g., expression or stability). With the aim of minimizing undesired effects of full-length GFP, here we describe the utility of a split GFP reporter during precrystallization studies of membrane proteins. GFP fluorescence appeared by complementation of the first 15 residues of GFP (GFP(11)) (fused to the C terminus of a membrane protein target) with the remaining nonfluorescent GFP (GFP(1-10)). The signal obtained after sequential expression of SteT (l-serine/l-threonine exchanger of Bacillus subtilis) fused to GFP(11) followed by GFP(1-10) specifically measured the protein fraction inserted into the Escherichia coli cytoplasmic membrane, thereby discarding protein aggregates confined as inclusion bodies. Furthermore, in vitro complementation of purified SteT-GFP(11) with purified GFP(1-10) was exploited to rapidly assess the stability of wild-type and G294V mutant versions of SteT-GFP(11) following detergent solubilization and purification. This method can be applied in a medium- to high-throughput manner with multiple samples.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Membrana/biossíntese , Proteínas de Bactérias/genética , Cristalografia por Raios X , Detergentes/química , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana/genética , Estabilidade Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
16.
Proc Natl Acad Sci U S A ; 108(10): 3935-40, 2011 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-21368142

RESUMO

Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na(+)-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.


Assuntos
Aminoácidos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica
17.
J Biol Chem ; 285(37): 28764-76, 2010 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-20610400

RESUMO

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same alpha-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC(50) similar to the apparent K(M) of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Substituição de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Transporte Biológico/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagênese , Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
18.
J Biol Chem ; 280(9): 7487-92, 2005 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-15623511

RESUMO

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu-14) from both EmrE monomers. Carbodiimide modification of EmrE has been studied using functional assays, and the evidence suggests that Glu-14 is the target of the reaction. Here we exploited electrospray ionization mass spectrometry to directly monitor the reaction with each monomer rather than following inactivation of the functional unit. A cyanogen bromide peptide containing Glu-14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2NPYIYLGGAILAEVIGTTLM(21) is approximately 80% modified in a time-dependent fashion, indicating that each Glu-14 residue in the oligomer is accessible to DiPC. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of Glu-14 with DiPC by up to 80%. Taken together with other biochemical data, the findings support a "time sharing" mechanism in which both Glu-14 residues in a dimer are involved in tetraphenylphosphonium and H(+) binding.


Assuntos
Antiporters/química , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Antiporters/ultraestrutura , Sítios de Ligação , Transporte Biológico , Carbodi-Imidas/farmacologia , Brometo de Cianogênio/química , Dimerização , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Ácido Glutâmico/química , Concentração de Íons de Hidrogênio , Proteínas de Membrana/ultraestrutura , Modelos Biológicos , Modelos Químicos , Oniocompostos/química , Compostos Organofosforados/química , Peptídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Prótons , Espectrometria de Massas por Ionização por Electrospray , Fatores de Tempo
19.
J Biol Chem ; 279(47): 49214-21, 2004 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-15364943

RESUMO

Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.


Assuntos
Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/biossíntese , Sítios de Ligação , Transporte Biológico , Bromosuccinimida/farmacologia , Cristalografia por Raios X , Escherichia coli/genética , Ácido Glutâmico/química , Ligação de Hidrogênio , Cinética , Espectrometria de Massas , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Modelos Moleculares , Mutação , Oxigênio/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Espectrofotometria , Especificidade por Substrato , Fatores de Tempo , Triptofano/química
20.
Proc Natl Acad Sci U S A ; 100(22): 12706-11, 2003 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-14566061

RESUMO

The crystal structure of the Escherichia coli lactose permease at 3.5 A with a bound substrate has been reported recently. The structure reveals the sugar-protein contacts, which include hydrophobic stacking between the galactopyranosyl ring of substrate and the indole side chain of Trp-151, as proposed previously. The nature of this interaction is studied here by exploiting the luminescence properties of Trp-151 in a mutant devoid of other tryptophan residues. The following phenomena are observed. (i) The fluorescence emission spectrum of Trp-151 and fluorescence-quenching experiments with water-soluble quenchers demonstrate that Trp-151 is in a hydrophilic environment. (ii) Substrate binding leads to a blue shift in the emission spectrum and reduction in accessibility to polar quenchers, indicating that Trp-151 becomes less exposed to aqueous solvent. (iii) The phosphorescence spectrum of Trp-151 is red-shifted in the presence of substrate, indicating charge separation of the triplet state due to a direct stacking interaction between the galactopyranosyl and indole rings. The spectroscopic data fully complement the x-ray structure and demonstrate the feasibility of fluorescence spectroscopy for studying sugar-protein interactions.


Assuntos
Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos , Simportadores , Triptofano/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Cinética , Medições Luminescentes , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Espectrometria de Fluorescência
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