RESUMO
Chitinase chi18-5 is an enzyme able to hydrolyze chitin and chitosan producing chitooligosaccharides (COS) of potential technological interest. chi18-5 is produced naturally by the fungus Trichoderma atroviride. It belongs to the glycosyl hydrolase (GH) family 18 of the Carbohydrate Active Enzyme (CAZy) database and it has 83% identity compared to the well-characterized chi42 of Trichoderma harzianum. Several efforts have been made to characterize the biochemical activity of the enzyme and its structure. Here, we studied the biophysical properties of recombinant chi18-5. In order to gain insight into its structure and stability, we studied thermal denaturation by Circular Dichroism (CD), Intrinsic Fluorescence (FL), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) at several pH between 3 and 8. We observed that the conformation of chi18-5 changes near its pI, and the transitions as a function of the temperature involved an increment in ß-sheet secondary structure at the expenses of âº-helix. We also performed amide hydrogen exchange dynamics in selected conditions. At pH ≤ 6, the proportion of fast exchanging residues are larger than at pH ≥ 6. Our results suggest that at pH below pI, chi18-5 is in a less compact structure which may have influence in the interaction with substrate and enzyme activity. KEY POINTS: ⢠Characterization of enzyme behavior is critical for their wide applications ⢠We produced and characterized biophysically a chitinase as a function of pH ⢠The pH of optimum activity correlates with a less compact structure of chi18-5.
Assuntos
Quitinases , Quitina , Quitinases/genética , Quitinases/metabolismo , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
We studied the conformational changes of the fatty acid-binding protein ReP1-NCXSQ in the interface of anionic lipid membranes. ReP1-NCXSQ is an acidic protein that regulates the activity of the Na+/Ca2+ exchanger in squid axon. The structure is a flattened barrel composed of two orthogonal ß-sheets delimiting an inner cavity and a domain of two α-helix segments arranged as a hairpin. FTIR and CD spectroscopy showed that the interactions with several anionic lipids in the form of small unilamellar vesicles (SUVs) induced an increase in the proportion of helix secondary structure. Lower amount or no increase in α-helix was observed upon the interaction with anionic lipids in the form of large unilamellar vesicles (LUVs). The exception was 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) that was equally efficien to to induce the conformational change both in SUVs and in LUVs. In solution, the infrared spectra of ReP1-NCXSQ at temperatures above the unfolding displayed a band at 1617 cm-1 characteristic of aggregated strands. This band was not observed when the protein interacted with DMPG, indicating inhibition of aggregation in the interface. Similarly to the observed in L-BABP, another member of the fatty acid binding proteins, a conformational change in ReP1-NCXSQ was coupled to the gel to liquid-crystalline lipid phase transition.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Transição de Fase , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Trocador de Sódio e Cálcio/metabolismoRESUMO
The regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ) is a 15kDa soluble, intracellular protein that regulates the activity of the Na(+)/Ca(2+) exchanger in the squid axon. It is a member of the cellular retinoic acid-binding proteins family and the fatty acid-binding proteins superfamily. It is composed of ten beta strands defining an inner cavity and a domain of two short alpha helix segments. In this work, we studied the binding and orientation of ReP1-NCXSQ in anionic and zwitterionic lipid membranes using molecular dynamics (MD) simulations. Binding to lipid membranes was also measured by filtration binding assay. ReP1-NCXSQ acquired an orientation in the anionic membranes with the positive end of the macrodipole pointing to the lipid membrane. Potential of mean force calculations, in agreement with experimental measurements, showed that the binding to the anionic interfaces in low ionic strength was stronger than the binding to anionic interfaces in high ionic strength or to zwitterionic membranes. The results of MD showed that the electrostatic binding can be mediated not only by defined patches or domains of basic residues but also by a global asymmetric distribution of charges. A combination of dipole-electric field interaction and local interactions determined the orientation of ReP1-NCXSQ in the interface.
Assuntos
Eletricidade , Proteínas de Ligação a Ácido Graxo/metabolismo , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Decapodiformes , Proteínas de Ligação a Ácido Graxo/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Conformação Proteica , Trocador de Sódio e Cálcio/químicaRESUMO
Phloretin is a known modifier of the internal dipole potential of lipid membranes. We studied the interaction of phloretin with model lipid membranes and how it influences the membrane dipole organization using ANS as fluorescent probe. The fluorescence increase observed when ANS binds to DMPC liposomes in gel phase (13 °C) was 2.5 times larger in the presence of phloretin. This effect was due to an increase in ANS affinity, which can be related to the known capability of phloretin in decreasing the dipole potential. Conversely, when the experiments were carried out at 33 °C (liquid crystalline phase), phloretin completely inhibited the increase in ANS fluorescence. In addition, phloretin only affected the electrical properties of the membrane in the gel phase, whereas it modifies structural ones in the liquid-crystalline state. We postulate that phloretin was bound only to the DMPC interface in the gel phase decreasing the surface negative charge density without modifying the structural properties of the ANS binding sites. In the liquid-crystalline phase instead, it increased the accessibility of water to the ANS binding sites decreasing the intrinsic affinity and the fluorescence quantum yield of ANS.
Assuntos
Naftalenossulfonato de Anilina/química , Dimiristoilfosfatidilcolina/química , Géis/química , Cristais Líquidos/química , Floretina/química , Lipossomos/químicaRESUMO
The weak hydrophobic acid carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) is a protonophoric uncoupler of oxidative phosphorylation in mitochondria. It dissipates the electrochemical proton gradient (ΔµH (+)) increasing the mitochondrial oxygen consumption. However, at concentrations higher than 1 µM it exhibits additional effects on mitochondrial energy metabolism, which were tentatively related to modifications of electrical properties of the membrane. Here we describe the effect of FCCP on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. FCCP inhibited the binding of ANS to liposomes either in the gel or in the liquid crystalline phase, by increasing the apparent dissociation constant of ANS. Smaller effect on the dissociation constant was observed at high ionic strength, suggesting that the effect of FCCP is through modification of the electrostatic properties of the membrane interface. In addition, FCCP also decreased (approximately 50 %) the quantum yield and increased the intrinsic dissociation constant of membrane-bound ANS, results that suggest that FCCP makes the environment of the ANS binding sites more polar. On those grounds we postulate that the binding of FCCP: i) increases the density of negative charges in the membrane surface; and ii) distorts the phospholipid bilayer, increasing the mobility of the polar headgroups making the ANS binding site more accessible to water.
Assuntos
Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/química , Lipossomos/química , Fosfatidilcolinas/química , Sítios de Ligação , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Fosforilação Oxidativa , Fosfatidilcolinas/metabolismoRESUMO
A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a â¼ 100-fold lower turnover number compared to the wild type enzyme. UV-Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.
Assuntos
Cobre , Sinorhizobium meliloti , Cobre/química , Nitritos/química , Sinorhizobium meliloti/química , Sinorhizobium meliloti/metabolismo , Histidina/química , Domínio Catalítico , Oxirredução , Ligantes , Glicina , Espectroscopia de Ressonância de Spin Eletrônica , Nitrito Redutases/químicaRESUMO
We studied the conformation of ß2-human glycoprotein (ß2GPI) in solution and bound to the anionic lipids palmitoyl oleoyl phosphatidylglycerol (POPG), dimiristoyl phosphatidylglycerol (DMPG) and dipalmitoyl phosphatidylglycerol (DPPG) as a function of the temperature. We used the infrared amide I' band to study the protein conformation, and the position of the antisymmetric stretching band of the methylene groups in the lipid hydrocarbon chains to study the lipid order. Lipid-protein complexes were studied in media of low and high ionic strengths. In solution, ß2GPI displayed a conformational pre-transition in the range 47-50°C, characterized by a shift in the band of ß secondary structure, previous to the main unfolding at 64°C. When the protein was bound to the anionic lipid membranes at 25°C, a similar shift as in the pre-transition in solution was observed, together with an increase in the band corresponding to α-helix secondary structure. Lipid-protein complexes formed large aggregates within the temperature range 10â 60°C. At temperatures above the protein unfolding, the complexes were disrupted to yield vesicles with bound protein. This finding indicated that the native fold was required for the formation of the lipid-protein aggregates. Cycles of heating and cooling showed hysteresis in the formation of aggregates.
Assuntos
Glicoproteínas/química , Lipídeos/química , Fosfatidilgliceróis/química , Calorimetria/métodos , Luz , Nefelometria e Turbidimetria , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Espalhamento de Radiação , Espectrofotometria/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , TemperaturaRESUMO
MutS initiates mismatch repair by recognizing mismatches in newly replicated DNA. Specific interactions between MutS and mismatches within double-stranded DNA promote ADP-ATP exchange and a conformational change into a sliding clamp. Here, we demonstrated that MutS from Pseudomonas aeruginosa associates with primed DNA replication intermediates. The predicted structure of this MutS-DNA complex revealed a new DNA binding site, in which Asn 279 and Arg 272 appeared to directly interact with the 3'-OH terminus of primed DNA. Mutation of these residues resulted in a noticeable defect in the interaction of MutS with primed DNA substrates. Remarkably, MutS interaction with a mismatch within primed DNA induced a compaction of the protein structure and impaired the formation of an ATP-bound sliding clamp. Our findings reveal a novel DNA binding mode, conformational change and intramolecular signaling for MutS recognition of mismatches within primed DNA structures.
Assuntos
Proteínas de Escherichia coli , Proteína MutS de Ligação de DNA com Erro de Pareamento , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases , DNA/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Ligação ProteicaRESUMO
Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1(D198A)) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1(D189A) also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.
Assuntos
Retículo Endoplasmático/enzimologia , Galactosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Cricetinae , Cricetulus , Retículo Endoplasmático/genética , Galactosiltransferases/genética , Complexo de Golgi/genética , Isoquinolinas/farmacologia , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
There are several examples of single mutations that lead to a well-defined disease through a well-known mechanism. In other cases, a collection of mutations of the same protein produces a pathology with different degrees of severity. The accompanying work by Uusitalo et al. studies several mutants of the fatty acid binding protein P2 of the peripheral nervous system myelin. They conserve the native tertiary structure but a remarkable difference in the capacity to interact with lipids. This could be a clue to unravel the complex way in which these mutations affect myelin structure and function in a variant of Charcot-Marie-Tooth disease. Comment on: https://doi.org/10.1111/febs.16079.
Assuntos
Doença de Charcot-Marie-Tooth , Doença de Charcot-Marie-Tooth/genética , Humanos , Mutação , Bainha de Mielina , ProteínasRESUMO
ß2 glycoprotein I (ß2GPI) is a soluble protein that participates in blood coagulation, clearance of apoptotic bodies and generation of antigens in antiphospholipid syndrome among many other functions. We studied the aggregates formed by ß2GPI with the anionic phospholipids palmitoyloleoylphosphatidyl glycerol, dimyristoylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol and cardiolipin using small angle X-ray scattering. The complexes obtained in a medium containing 0.01 M NaCl showed Bragg peaks up to the sixth order in a well-defined integer sequence indicating the presence of a lamellar stacking with a periodicity of 17.8 nm and with largely reduced membrane fluctuations. Modeling the complex signal allowed us to conclude that the coherence length was only two bilayers and that about 15% of the total surface was actually stacked. The space between bilayers allows accommodating an extended ß2GPI molecule making a bridge between the interacting bilayers. The interactions between membranes mediated by ß2GPI was favored when the membranes were in the liquid crystalline state.
Assuntos
Síndrome Antifosfolipídica , Cardiolipinas , Humanos , Membranas , Fosfolipídeos , beta 2-Glicoproteína IRESUMO
The aim of this study was to design and develop a novel hybrid formulation based on lipid nanocapsules containing bevacizumab (BVZ), an effective therapeutic antibody, on the surface and triamcinolone acetonide (TA) in the inner core (BVZ-TA-LNC) intended to improve ocular therapy. Hence, a phase inversion-insertion one step method was developed to drug loading and surface modification of lipid nanocapsules by post-insertion of a bifunctional polymer, followed by antibody coupling using "click" chemistry. The covalent bond and antibody capacity binding to its specific antigen were confirmed by thermal analysis and immunoassay, respectively. BVZ-TA-LNC presented nanometric size (102 nm), negative surface potential (-19 mV) and exhibiting 56% of TA in the lipid core. BVZ-TA-LNC tended to prevent the endothelial cell migration and significantly prevented the capillary formation induced by the vascular endothelium growth factor (VEGF). The novel hybrid system allowed the co-loading of two different therapeutic molecules and may be promising to improve the therapy of eye disorders that occur with inflammation and/or neovascularization.
Assuntos
Nanocápsulas , Triancinolona Acetonida , Anticorpos Monoclonais , Bevacizumab/uso terapêutico , LipídeosRESUMO
Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na(+)/Ca(2+) exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far-UV circular dichroism and infrared spectra suggest a majority of beta-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten beta-sheets and two short alpha-helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.
Assuntos
Trifosfato de Adenosina/farmacologia , Decapodiformes/fisiologia , Gânglios/fisiologia , Neurônios/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Regiões 5' não Traduzidas/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Cinética , Dados de Sequência Molecular , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Espectrofotometria Infravermelho , Raios UltravioletaRESUMO
Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a beta-barrel of ten strands capped with a short helix-loop-helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.
Assuntos
Proteínas de Transporte/química , Membrana Celular/química , Simulação por Computador , Glicoproteínas de Membrana/química , Modelos Químicos , Fosfolipídeos/química , Motivos de Aminoácidos/fisiologia , Animais , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Galinhas , Glicoproteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica/fisiologiaRESUMO
We designed an experimental approach to differentiate the kinetics of protein binding to a lipid membrane from the kinetics of the associated conformational change in the protein. We measured the fluorescence intensity of the single Trp6 in chicken liver bile acid-binding protein (L-BABP) as a function of time after mixing the protein with lipid membranes. We mixed the protein with pure lipid membranes, with lipid membranes in the presence of a soluble quencher, and with lipid membranes containing a fluorescence quencher attached to the lipid polar head group. We fitted simultaneously the experimental curves to a three-state kinetic model. We conclude that in a first step, the binding of L-BABP to the interfacial region of the anionic lipid polar head groups occurred simultaneously with a conformational change to the partly unfolded state. In a second slower step, Trp6 buried within the polar head group region, releasing contacts with the aqueous phase.
Assuntos
Proteínas de Transporte/química , Glicoproteínas de Membrana/química , Modelos Químicos , Triptofano/química , Lipossomas Unilamelares/química , Animais , Galinhas , Fluorescência , Cinética , Ligação Proteica , Conformação Proteica , Dobramento de ProteínaRESUMO
Interactions between the antibiotic peptide nisin and multilamellar vesicles of phosphoglycerol lipids in different phase-states were studied using vibrational spectroscopy. The infrared amide I' band of nisin, both in solution and in the membrane-bound state, was analyzed in the temperature range comprised between 20 and 60⯰C in order to study its conformational behavior. Nisin presented mainly unordered and ß-turns conformations. Their relative populations varied according to the environment and as the temperature increased: ß turns were more favored in the membrane-bound state than in solution, but at higher temperatures the disordered conformation was dominant in both states. Spectral changes of specific infrared bands belonging to the hydrocarbon and polar moieties of lipids were also analyzed to evaluate the perturbation of the lipid membrane order. Nisin interactions with the membrane polar region induced a high restriction to water incorporation, promoting a small increase in the temperature of the lipid phase transition. Raman spectra of nisin/phosphoglycerol systems at ambient temperature were also analyzed. They revealed that the peptide incorporation to a membrane in the fluid phase caused drastic structural modifications in the hydrophobic region of the bilayer. Although nisin may be able to disrupt the hydrophobic portion of the bilayer in the gel phase, the most of the peptide molecule remained at the membrane surface interacting with the polar headgroups. This work provides evidence of a differential effect of nisin on anionic membranes, depending on the phase-state of the lipid.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Nisina/química , Nisina/metabolismo , Análise Espectral Raman/métodos , Ânions , Modelos Biológicos , VibraçãoRESUMO
Calreticulin (CRT) is a calcium-binding protein that participates in several cellular processes including the control of protein folding and homeostasis of Ca2+. Its folding, stability and functions are strongly controlled by the presence of Ca2+. The oligomerization state of CRT is also relevant for its functions. We studied the thermal transitions of monomers and oligomers of CRT by differential scanning calorimetry (DSC), circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) in the presence and absence of Ca2+. We found three and two components for the calorimetric transition in the presence and absence of Ca2+ respectively. The presence of several components was also supported by CD and FTIR spectra acquired as a function of the temperature. The difference between the heat capacity of the native and the unfolded state strongly suggests that interactions between protein domains also contribute to the heat uptake in a calorimetry experiment. We found that once unfolded at high temperature the process is reversible and the native state can be recovered upon cooling only in the absence of Ca2+. We also propose a new simple method to obtain pure CRT oligomers.
Assuntos
Calreticulina/química , Cálcio/química , Varredura Diferencial de Calorimetria , Calreticulina/genética , Dicroísmo Circular , Conformação Proteica , Desdobramento de Proteína , Proteínas Recombinantes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , TermodinâmicaRESUMO
Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98-106]. We studied the infrared amide I' band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 degrees C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 degrees C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 degrees C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Galinhas/genética , Bicamadas Lipídicas/metabolismo , Fígado/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Transição de Fase , Animais , Varredura Diferencial de Calorimetria , Fosfatidilgliceróis/metabolismo , Conformação Proteica , Dobramento de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , TemperaturaRESUMO
The fatty acid-binding proteins L-BABP and Rep1-NCXSQ bind to anionic lipid membranes by electrostatic interactions. According to Molecular Dynamics (MD) simulations, the interaction of the protein macrodipole with the membrane electric field is a driving force for protein binding and orientation in the interface. To further explore this hypothesis, we studied the interactions of these proteins with cationic lipid membranes. As in the case of anionic lipid membranes, we found that both proteins, carrying a negative as well as a positive net charge, were bound to the positively charged membrane. Their major axis, those connecting the bottom of the ß-barrel with the α-helix portal domain, were rotated about 180 degrees as compared with their orientations in the anionic lipid membranes. Fourier transform infrared (FTIR) spectroscopy of the proteins showed that the positively charged membranes were also able to induce conformational changes with a reduction of the ß-strand proportion and an increase in α-helix secondary structure. Fatty acid-binding proteins (FABPs) are involved in several cell processes, such as maintaining lipid homeostasis in cells. They transport hydrophobic molecules in aqueous medium and deliver them into lipid membranes. Therefore, the interfacial orientation and conformation, both shown herein to be electrostatically determined, have a strong correlation with the specific mechanism by which each particular FABP exerts its biological function.
Assuntos
Membrana Celular/química , Proteínas de Ligação a Ácido Graxo/química , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Membrana Celular/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Estrutura Secundária de Proteína , Eletricidade EstáticaRESUMO
Line tension (γ) is a key parameter for the structure and dynamics of membrane domains. It was proposed that hybrid lipids, with mixed saturated and unsaturated acyl chains, participate in the relaxation of γ through different mechanisms. In this work, we used molecular dynamics simulations of the coarse-grained MARTINI model to measure γ in liquid-ordered-liquid-disordered (Lo-Ld) membranes, with increasingly larger relative proportion of the hybrid polyunsaturated lipid PAPC (4:0-5:4PC) to DAPC (di5:4PC) (i.e., XH). We also calculated an elastic contribution to γ by the Lo-Ld thickness mismatch, tilt moduli, and bending moduli, as predicted by theory. We found that an increase in XH decreased the overall γ value and the elastic contribution to line tension. The effect on the elastic line tension is driven by a reduced hydrophobic mismatch. Changes in the elastic constants of the phases due to an increase in XH produced a slightly larger elastic γ term. In addition to this elastic energy, other major contributions to γ are found in these model membranes. Increasing XH decreases both elastic and nonelastic contributions to γ. Finally, PAPC also behaves as a linactant, relaxing γ through an interfacial effect, as predicted by theoretical results. This study gives insight into the actual contribution of distinct energy terms to γ in bilayers containing polyunsaturated hybrid lipids.