RESUMO
The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.
Assuntos
Bicamadas Lipídicas/química , Peptídeos/química , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Proteínas de Membrana/química , Dados de Sequência Molecular , Fosfolipídeos/química , Conformação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , TermodinâmicaRESUMO
In this work, we performed targeted immobilization of immunoglobulins by means of bacterial S-layer proteins from Bacillus coagulans E38-66/V1 recrystallized on liposomes, which were exploited as immobilization matrix for antibody (Ab)-human IgG. The study of interaction of rabbit or swine anti-human IgG as antigens (Ag) was performed by means of measuring changes of ultrasound velocity. We showed that at a temperature of 25 degrees C, the increment of ultrasound velocity [u] linearly decreased following an increase of concentration of Ag. The decrease of [u] was presumably due to changes of hydration of the membrane due to the binding process. Approximately 10 times lower changes of [u] were observed at 45 degrees C for Ag-Ab interaction as well as for nonspecific interaction of Ag with liposomes covered by S-layer without Ab. No substantial differences in the behaviour of [u] were observed for interactions of human IgG with rabbit or swine anti-human IgG.
Assuntos
Proteínas de Bactérias/metabolismo , Imunoglobulina G/metabolismo , Lipossomos , Ultrassom , Densitometria , HumanosRESUMO
We tested the possibility of amperometric detection of DNA hybridization on a gold surface influenced by the immobilization of oligonucleotide giving different orientations of single stranded DNA relative to the gold surface. The DNA sensor was fabricated by chemisorption of 18-mer oligonucleotide modified by a phosphorothioate group either at its 3' or both 3' and 5' terminal. After immobilization of oligonucleotide to the gold support, the sensor was immersed in 11-mercaptoundecanoic acid (MUA) solution. Further chemisorption of MUA resulted in approximately 10-fold increase of resistance of the organic layer. Addition of complementary oligonucleotide resulted in an increase of conductivity for DNA sensor oriented perpendicular to the gold support (DNA with one thiol group), while the conductance decreased for DNA sensor with single stranded DNA oriented parallel to the gold support (with DNA modified by thiol groups at both 3' and 5' terminals). Addition of non-complementary chain resulted a slight decrease or no change of sensor conductivity. The hybridization process at both types of DNA orientations is not cooperative and can be described by Langmuir isotherms. The hybridization event on gold support has been confirmed by mass detection using the quartz crystal microbalance technique.
Assuntos
DNA/química , Ouro/química , Conformação de Ácido Nucleico , Sequência de Bases , EletroquímicaRESUMO
We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.
Assuntos
Antibacterianos/farmacologia , Gramicidina/farmacologia , Bicamadas Lipídicas/química , Densitometria , Dimiristoilfosfatidilcolina/química , Estrutura Molecular , Conformação Proteica , Temperatura , Termodinâmica , UltrassomRESUMO
Various methods have been applied to study the physical properties of metal supported bilayer lipid membranes (s-BLM) and large unilamellar liposomes containing biotinylated phospholipids during the binding of IgG modified by avidin. Electrostriction method applied to s-BLM showed that addition of avidin-IgG (A-IgG) complex in a small concentration (0.2 mumol/l) resulted in an approximately twofold decrease of membrane capacitance, C, increase of elasticity modulus in direction perpendicular to the membrane plane, E perpendicular, by 5-20%, increase in intrinsic membrane potential, delta phi m, by approximately 30 mV, and an approximately 5-15% increase of the coefficient of dynamic viscosity, eta. The method of ultrasonic velocimetry showed that addition into the suspension of liposomes of unmodified IgG did not induce any changes in ultrasound velocity, while addition of A-IgG complex in a concentration range of 0-1 mumol/l led to an increase of the velocity number [u]. The plot of changes of [u] versus A-IgG concentration has a tendency to saturate at concentrations above 0.5 mumol/l, which suggests long-range interactions in the membrane induced by strong binding of A-IgG to the biotin sites on the membrane. Probably, this binding leads to a decrease of the coefficient of adiabatic compressibility of liposomes.
Assuntos
Avidina/química , Imunoglobulina G/química , Bicamadas Lipídicas/química , Lipossomos/química , Elasticidade , Potenciais da Membrana , ViscosidadeRESUMO
The sound velocity and density of suspensions of large unilamellar liposomes from dimyristoylphosphatidylcholine with admixed cholesterol have been measured as a function of temperature around the chain melting temperature of the phospholipid. The cholesterol-to-phospholipid molar ratio xc has been varied over a wide range (0 = xc = 0.5). The temperature dependence of the sound velocity number, of the apparent specific partial volume of the phospholipid, and of the apparent specific adiabatic compressibility have been derived from the measured data. These data are particularly discussed with respect to the volume fluctuations within the samples. A theoretical relation between the compressibility and the excess heat capacity of the bilayer system has been derived. Comparison of the compressibilities (and sound velocity numbers) with heat capacity traces display the close correlation between these quantities for bilayer systems. This correlation appears to be very useful as it allows some of the mechanical properties of membrane systems to be calculated from the specific heat capacity data and vice versa.