RESUMEN
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.
Asunto(s)
Cobre , Sinorhizobium meliloti , Cobre/química , Nitritos/química , Sinorhizobium meliloti/química , Sinorhizobium meliloti/metabolismo , Histidina/química , Dominio Catalítico , Oxidación-Reducción , Ligandos , Glicina , Espectroscopía de Resonancia por Spin del Electrón , Nitrito Reductasas/químicaRESUMEN
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.
Asunto(s)
Síndrome Antifosfolípido , Cardiolipinas , Humanos , Membranas , Fosfolípidos , beta 2 Glicoproteína IRESUMEN
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.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Enfermedad de Charcot-Marie-Tooth/genética , Humanos , Mutación , Vaina de Mielina , ProteínasRESUMEN
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.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Nisina/química , Nisina/metabolismo , Espectrometría Raman/métodos , Aniones , Modelos Biológicos , VibraciónRESUMEN
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.
Asunto(s)
Calreticulina/química , Calcio/química , Rastreo Diferencial de Calorimetría , Calreticulina/genética , Dicroismo Circular , Conformación Proteica , Desplegamiento Proteico , Proteínas Recombinantes/química , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , TermodinámicaRESUMEN
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.
Asunto(s)
Membrana Celular/química , Proteínas de Unión a Ácidos Grasos/química , Lípidos de la Membrana/química , Simulación de Dinámica Molecular , Membrana Celular/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
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.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Transición de Fase , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
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.
Asunto(s)
Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Fosfatidilcolinas/química , Colesterol/química , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , TemperaturaRESUMEN
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.
Asunto(s)
Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/química , Liposomas/química , Fosfatidilcolinas/química , Sitios de Unión , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Fosforilación Oxidativa , Fosfatidilcolinas/metabolismoRESUMEN
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.
Asunto(s)
Electricidad , Proteínas de Unión a Ácidos Grasos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Decapodiformes , Proteínas de Unión a Ácidos Grasos/química , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Simulación de Dinámica Molecular , Conformación Proteica , Intercambiador de Sodio-Calcio/químicaRESUMEN
Protein arginylation mediated by arginyl-tRNA protein transferase is a post-translational modification that occurs widely in biology, it has been shown to regulate protein and properties and functions. Post-translational arginylation is critical for embryogenesis, cardiovascular development and angiogenesis but the molecular effects of proteins arginylated in vivo are largely unknown. In the present study, we demonstrate that arginylation reduces CRT (calreticulin) thermostability and induces a greater degree of dimerization and oligomerization. R-CRT (arginylated calreticulin) forms disulfide-bridged dimers that are increased in low Ca(2+) conditions at physiological temperatures, a similar condition to the cellular environment that it required for arginylation of CRT. Moreover, R-CRT self-oligomerizes through non-covalent interactions that are enhanced at temperatures above 40 °C, condition that mimics the heat shock treatment where R-CRT is the only isoespecies of CRT that associates in cells to SGs (stress granules). We show that in cells lacking CRT the scaffolding of larger SGs is impaired; the transfection with CRT (hence R-CRT expression) restores SGs assembly whereas the transfection with CRT mutated in Cys146 does not. Thus, R-CRT disulfide-bridged dimers (through Cys146) are essential for the scaffolding of larger SGs under heat shock, although these dimers are not required for R-CRT association to SGs. The alteration in SGs assembly is critical for the normal cellular recover of cells after heat induced stress. We conclude that R-CRT is emerging as a novel protein that has an impact on the regulation of SGs scaffolding and cell survival.
Asunto(s)
Arginina/química , Calreticulina/química , Calreticulina/metabolismo , Proteínas de Choque Térmico/metabolismo , Aminoaciltransferasas , Animales , Apoptosis , Línea Celular , Gránulos Citoplasmáticos/metabolismo , Dimerización , Respuesta al Choque Térmico , Ratones , Procesamiento Proteico-PostraduccionalRESUMEN
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.
Asunto(s)
Glicoproteínas/química , Lípidos/química , Fosfatidilgliceroles/química , Calorimetría/métodos , Luz , Nefelometría y Turbidimetría , Conformación Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Dispersión de Radiación , Espectrofotometría/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , TemperaturaRESUMEN
Avian liver bile acid-binding protein (L-BABP) binds peripherically to anionic lipid membranes. We previously showed that in the absence of added salt the binding to 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) occurs with changes in the secondary structure, the extent of which depends on the phase state of the lipid. In the present work, we used Fourier transform infrared spectroscopy to study the conformations of L-BABP bound to lipids with phosphoglycerol and phosphatidic acid polar head groups and with different transition temperatures in an aqueous medium with high ionic strength (0.1 M NaCl). When L-BABP was bound to the lipids with saturated acyl chains, DMPG, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), and 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA), the conformation shifted from a native-like secondary structure to an unfolded state at the temperature of lipid chain melting. The protein was in the native-like conformation when it was bound to the unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in the liquid-crystalline phase. We also measured the electrokinetic surface potential of POPG and DMPG vesicles in the gel and in the liquid-crystalline phase and the protein binding constant to these lipid membranes. We found a correlation indicating that protein unfolding in the interface was due to the increase in the electrostatic surface potential that occurs in the lipid phase transition.
Asunto(s)
Proteínas Portadoras/química , Membrana Dobles de Lípidos/química , Glicoproteínas de Membrana/química , Lípidos de la Membrana/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Animales , Aves , Transición de Fase , Unión Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad EstáticaRESUMEN
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.
Asunto(s)
Proteínas Portadoras/química , Glicoproteínas de Membrana/química , Modelos Químicos , Triptófano/química , Liposomas Unilamelares/química , Animales , Pollos , Fluorescencia , Cinética , Unión Proteica , Conformación Proteica , Pliegue de ProteínaRESUMEN
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.
Asunto(s)
Proteínas Portadoras/química , Membrana Celular/química , Simulación por Computador , Glicoproteínas de Membrana/química , Modelos Químicos , Fosfolípidos/química , Secuencias de Aminoácidos/fisiología , Animales , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Pollos , Glicoproteínas de Membrana/metabolismo , Fosfolípidos/metabolismo , Unión Proteica/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Pollos/genética , Membrana Dobles de Lípidos/metabolismo , Hígado/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Transición de Fase , Animales , Rastreo Diferencial de Calorimetría , Fosfatidilgliceroles/metabolismo , Conformación Proteica , Pliegue de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad Estática , TemperaturaRESUMEN
We have studied the thermodynamic, surface, and structural properties of alphaM1 transmembrane sequence of the nicotinic acetylcholine receptor (nAChR) by using Langmuir monolayer, FT-IR spectroscopy and molecular dynamics simulation techniques in membrane-mimicking environments. M1 spontaneously incorporates into a lipid-free air-water interface, showing a favourable adsorption free energy of -7.2 kcal/mol. A cross-sectional molecular area of 210 A(2)/molecule, a surface potential of 4.2 fV/molecule and a high stability of the film were deducted from pure M1 monolayers. FT-IR experiments and molecular dynamics simulations in membrane-mimicking environments (sodium-dodecyl-sulfate and CCl(4), respectively) indicate coexistence between helical and non-helical structures. Furthermore, mixed peptide-lipid monolayers and monolayer penetration experiments were performed in order to study the peptide-lipid interaction. Mixed with condensed lipids (dipalmitoyl-phosphocholine, and dipalmitoyl-phosphoglycerol), M1 shows immiscible/miscible behaviour at low/high peptide concentration, respectively. Conversely, a complete miscible peptide-lipid interface is observed with liquid-expanded lipids (palmitoyl-oleoyl-phosphocholine, and palmitoyl-oleoyl-phosphoglycerol). Peptide penetration experiments demonstrate that the M1 peptide preferentially interacts with zwitterionic phosphocholine interfaces.
Asunto(s)
Fragmentos de Péptidos/química , Receptores Nicotínicos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Adsorción , Secuencia de Aminoácidos , Membrana Celular/química , Simulación por Computador , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Conformación Proteica , Subunidades de Proteína/química , Dodecil Sulfato de Sodio/química , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Termodinámica , Agua/químicaRESUMEN
Glycans are key structures involved in biological processes such as cell attachment, migration, and invasion. Information coded on cell-surface glycans is frequently deciphered by proteins, as lectins, that recognize specific carbohydrate topology. Here, we describe the fine carbohydrate specificity of Euphorbia milii lectin (EML). Competitive assays using various sugars showed that GalNAc was the strongest inhibitor, and that the hydroxyl axial position of C4 and acetamido on C2 of GalNAc are critical points of EML recognition. A hydrophobic locus adjacent to GalNAc is also an important region for EML binding. Direct binding assays of EML revealed a stereochemical requirement for a structure adjacent to terminal GalNAc, showing that GalNAc residue is a necessary but not sufficient condition for EML interaction. The capacity of EML to bind epithelial tumor cells makes it a potentially useful tool for study of some over-expressed GalNAc glycoconjugates.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Euphorbia/química , Lectinas/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Modelos Moleculares , Datos de Secuencia Molecular , EstereoisomerismoRESUMEN
Chicken liver bile acid-binding protein (formerly known as chicken liver basic fatty acid-binding protein) binds to anionic lipid membranes acquiring a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B., and Montich, G. (2003) Biochim. Biophys. Acta 1611, 98-106]. To understand the mechanisms of its interactions with membranes, we have investigated the presence of partly folded states in solution. Using fluorescence spectroscopy of the single Trp residue, circular dichroism in the far- and near-UV, Fourier transform infrared spectroscopy, and size-exclusion chromatography, we found that L-BABP was partly unfolded at pH 2.5 and low ionic strength, retaining some of its secondary structure. Addition of 0.1 M NaCl at pH 2.5 or decreasing the pH to 1.5 produced a more compact partly folded state, with a partial increase of secondary structure and none of tertiary structure. Fluorescence emission spectra of this state indicate that the Trp residue is within an environment of low polarity, similar to the native state. This environment is not produced by the insertion of the Trp into soluble aggregates as revealed by size-exclusion chromatography, fluorescence anisotropy, and infrared spectroscopy. The presence of partly folded states under acidic conditions in solution suggests the possibility that membrane binding of L-BABP occurs via this state.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Hígado/química , Hígado/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Pliegue de Proteína , Ácidos/química , Animales , Pollos , Cromatografía en Gel , Dicroismo Circular , Concentración de Iones de Hidrógeno , Concentración Osmolar , Estructura Secundaria de Proteína , Soluciones , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , TermodinámicaRESUMEN
Ligand binding to proteins is a key process in cell biochemistry. The interaction usually induces modifications in the unfolding thermodynamic parameters of the macromolecule due to the coupling of unfolding and binding equilibria. In addition, these modifications can be attended by changes in protein structure and/or conformational flexibility induced by ligand binding. In this work, we have explored the effect of biotin binding on conformation and dynamic properties of avidin by using infrared spectroscopy including kinetics of hydrogen/deuterium exchange. Our results, along with previously thermodynamic published data, indicate a clear correlation between thermostability and protein compactness. In addition, our results also help to interpret the thermodynamic binding parameters of the exceptionally stable biotin:AVD complex.