Monovalent and Multivalent Binding of Streptavidin to Biotinylated Gramicidin Affects the Kinetic Properties of the Ion Channel

Biotin-avidin (or streptavidin) high affinity binding has been widely applied as a universal tool for basic research as well as diagnostic and therapeutic purposes. Here we studied the interaction of streptavidin with ionic channels formed by biotinylated gramicidin in planar bilayer lipid membranes (BLM) using the method of sensitized photoinactivation. As shown previously, the addition of streptavidin leads to a profound increase in the lifetime (tau) of gA5XB, a biotinylated analog of gramicidin A with a linker arm of five aminocaproyl groups (Rokitskaya et al. (2000) Biochemistry, 39, 13053-13058). The present study has revealed that the increase in tau is related to multivalent interaction of streptavidin with biotinylated gramicidin, i.e., to formation of a complex of streptavidin with several gramicidin channels, whereas binding of streptavidin to a single channel does not change the value of tau. A rather long linker arm attaching biotin to the C-terminus of gramicidin appeared to be required for the multivalent interaction of streptavidin with gramicidin channels, as the increase in tau was not observed with channels formed by gA2XB, the biotinylated gramicidin analog with a linker arm comprising only two aminocaproyl groups. However, the formation of a stoichiometric (1 : 1) complex of streptavidin with gA2XB apparently occurred. The multivalent interaction of streptavidin with gA5XB disappeared if biotinylated lipids were included into the diphytanoylphosphatidylcholine membrane. It is suggested that the slowing of gramicidin channel kinetics provoked by streptavidin binding is due to membrane-mediated elastic interactions between two neighboring channels.     

Effect of avidin on channel kinetics of biotinylated gramicidin

Membrane protein functioning basically depends on the supramolecular structure of the proteins which can be modulated by specific interactions with external ligands. The effect of a water-soluble protein bearing specific binding sites on the kinetics of ionic channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM) has been studied using three independent approaches: (1) sensitized photoinactivation, (2) single-channel, and (3) autocorrelation measurements of current fluctuations. As shown previously [Rokitskaya, T. I., et al. (1996) Biochim. Biophys. Acta 1275, 221], the time course of the flash-induced current decrease in most cases follows a single-exponential decay with an exponential factor (tau) that corresponds to the gA single-channel lifetime. Addition of avidin does not affect tau for gA channels, but causes a dramatic increase in tau for channels formed by gA5XB, a biotinylated analogue of gA. This effect is reversed by addition of an excess of biotin to the bathing solution. The average single-channel duration of gA5XB was about 3.6 s as revealed by single-channel recording of the BLM current. After prolonged incubation with avidin, a long-lasting open state of the gA5XB channel appeared which did not close for more than 10 min. The data on gA5XB photoinactivation kinetics and single-channel measurements were confirmed by analysis of the corresponding power spectra of the current fluctuations obtained in the control, in the presence of avidin, and after the addition of biotin. We infer that avidin produces a deceleration of gA5XB channel kinetics by motional restriction of gA5XB monomers and dimers upon the formation of avidin and gA5XB complexes, which would stabilize the channel state and thus increase the single-channel lifetime.     

Gating and Permeation in Ion Channels Formed by Gramicidin A and Its Dioxolane-linked Dimer in Na+ and Cs+ Solutions

The association of two gramicidin A (gA) peptides via H-bonds in lipid bilayers causes the formation of an ion channel that is selective for monovalent cations only. In this study, two gAs were covalently linked with a dioxolane group (SS dimer). Some functional properties of natural gA channels were compared to that synthetic dimer in Na⁺- or Cs⁺-containing solutions. The SS dimer remained in the open configuration most of the time, while natural gA channels had a relatively brief mean open time. Single channel conductances to Na⁺ (g _Na ) or Cs⁺ (g _Cs ) in the SS dimer were smaller than in natural gA. However, g _Na was considerably more attenuated than g _Cs . This probably results from a tight solvation of Na⁺ by the dioxolane linker in the SS channel. In Cs⁺ solutions, the SS had frequent closures. By contrast, in Na⁺ solutions the synthetic dimer remained essentially in the open state. The mean open times of SS channels in different solutions (T _open,Na > T _open,Cs > T _open,H ) were inversely proportional to the single channel conductances (g _H > g _Cs > g _Na ). This suggests that ion occupancy inside the pore stabilizes the open configuration of the gA dimer. The mean closed time of the SS dimer was longer in Cs⁺ than in H⁺ solutions. Possible mechanisms for these effects are discussed. Received: 17 September 1999/Revised: 21 December 1999     

Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (²H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous ²H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, ²H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.Abbreviations DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol - DPPC-d₆₂ 1,2-perdeuterodipalmitoyl-sn-glycero-3-phosphocholine - DPPC-d₄ 1,2-dipalmitoyl-sn-glycero-3-phospho-(, perdeutero)-choline     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance (²H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogens in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T₁ relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Monitoring of <Emphasis Type="Italic">E. coli</Emphasis> immobilization on modified gold electrode: A new bacteria-based glucose sensor

Electrochemical impedance spectroscopy (EIS) technique has proved to be an effective method for monitoring the immobilization of various bioactive species such as enzymes, DNA, whole cells, and so forth. In this work we describe the development of an electrochemical whole cell based biosensor. Biotinylated fluorescent E. coli are immobilized onto a cysteamine, Sulfo-NHS-LC-biotin, and avidin modified gold electrodes. Immobilized bacteria are clearly observed using confocal microscopy. Electrochemical measurements are based on the charge-transfer kinetics of [Fe (CN)₆]^3−/4− redox couple. The experimental impedance data were modelised with a computer. SAM assembly and the subsequent immobilization of bacteria on the gold bare electrodes greatly increased the electrontransfer resistance (R _et ) and reduced the constant phase element (CPE). It’s interesting to note, the hard immobilization of bacteria on the surface of electrode and do not remove during measurements. The effect of glucose addition was studied in the range of 10⁻⁷ μM to 10 μM. The relation between the evolution of R _et and D-glucose concentration was found to be linear for values ranging from 10⁻⁵ μM to 10⁻¹ μM and reached saturation for higher concentrations. Such biosensor could be applied to a more fundamental study of cell metabolism and drugs effect.     

Tandem gramicidin channels cross-linked by streptavidin

The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053-13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

Electrochemically platinized carbon paste enzyme electrodes: A new design of amperometric glucose biosensors

A platinized carbon paste prepared via electrodeposition had a preferential electrocatalytic action toward H₂O₂. Therefore, we have developed a new amperometric glucose biosensor based on the immobilization of glucose oxidase on to the electrochemically platinized carbon paste. The proposed biosensor is free of potential interferences due to its cathodic detection of glucose at the potential of 0.0 V (vs. Ag/AgCl). It also shows acceptable analytical performance in terms of linearity (6 × 10⁻⁵ to 1.2 × 10⁻² M, r = 0.998), detection limit (2 × 10⁻⁵ M), response time (20–30 s), reproducibility (RSD = 4.4%), and storage life (t _0.80 = 45 days). All these advantages of the biosensor raise potential possibilities for its medical or other biotechnical applications.     

Fluid or gel phase lipid bilayers to study peptide-membrane interactions?

Since many studies on peptide-membrane interactions are carried out only with fluid phase lipid bilayers (L _α -phase, absence of cholesterol) we have investigated whether this phase is really a suitable model for biological membranes. For this purpose the action of melittin on zwitterionic and negatively charged phospholipid bilayers, in the absence and presence of 30 mol% cholesterol, was investigated by solid state ³¹P-NMR. From the NMR point of view, it appears that systems composed of a single phospholipid best mimic the sterol-containing system a few degrees below the gel-to-fluid phase transition, i. e., in the rippled phase (P _β' ). It is then proposed that a relatively rigid membrane containing local defects, rather than a L _α -bilayer, is required as an appropriate model for natural membranes when probing the action of melittin. Such requirements might be crucial when studying peptide-lipid interactions. Received: 5 February 1996 / Accepted: 1 July 1996     

A mediator-free amperometric hydrogen peroxide biosensor based on HRP immobilized on a nano-Au/poly 2,6-pyridinediamine-coated electrode

A mediator-free amperometric hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) enzyme on colloidal Au modified platinum (Pt) wire electrode, which was modified by poly 2,6-pyridinediamine (pPA). The modified process was characterized by electrochemical impedance spectroscopy (EIS), and the electrochemical characteristics of the biosensor were studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The biosensor displayed an excellent electrocatalytical response to reduction of H₂O₂ without the aid of an electron mediator, the linear range was 4.2 × 10⁻⁷–1.5 × 10⁻³ mol/L (r = 0.9977), with a detection limit of 1.4 × 10⁻⁷ mol/L. Moreover, the performance and factors influencing the resulted biosensor were studied in detail. The studied biosensor exhibited permselectivity, good stability and good fabrication reproducibility.     

Amperometric hydrogen peroxide biosensor based on covalently immobilizing thionine as a mediator

A novel amperometric hydrogen peroxide biosensor based on the immobilization of hemoglobin on the 2,6-pyridinedicarboxylic acid (PDC) polymer, thionine and nano-Au was successfully fabricated. In this strategy, PDC polymer acted as the matrices to covalently immobilize the thionine, and then hemoglobin was successfully adsorbed on the nano-Au which was electro-deposited on to thionine modified electrode surface. The preparation process of modified electrode was characterized with electrochemical impedance spectroscopy and atomic force microscope. The analytical performance of proposed biosensor toward H₂O₂ was investigated by cyclic voltammetry and chronoamperometry. The resulted biosensor exhibited fast amperometric response (within 6 s) to H₂O₂, and linear range was from 9.1 μM to 5.0 mM with the detection limit of 2.6 μM (S/N = 3). The apparent Michaelis–Menten constant (K _M^app) was evaluated to be 3.2 mM. Furthermore, the resulted biosensor showed good stability and reproducibility.     

Methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl groups in large proteins

A set of sensitive methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl positions in large proteins is described. The developed methodology is applied to an 82-kDa enzyme Malate Synthase G. Complete alanine β and isoleucine γ2 ¹H–¹³C methyl chemical shift assignments could be obtained from the set of new methyl-detected ‘out-and-back’ 3D experiments. The described methodology for methyl assignments should be applicable to protein molecules within approximately 100-kDa molecular weight range irrespective of the labeling strategy chosen to produce selectively protonated Alaβ and Ileγ2 ¹³CH₃ sites on a deuterated background.     

Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Summary Enteropeptidase (enterokinase EC 3.4.21.9), catalyzing trypsinogen activation, exhibits unique properties for high efficiency hydrolysis of the polypeptide chain after the N-terminal tetraaspartyl-lysyl sequence. This makes it a convenient tool for the processing of fusion proteins containing this sequence. We found the enteropeptidase-catalysing degradation of some bioactive peptides: cattle hemoglobin beta-chain fragments Hb (2–8) (LTAEEKA) and Hb (1–9) (MLTAEEKAA), human angiotensin II (DRVYIHPF) (AT). Model peptides with truncated linker WDDRG and WDDKG also were shown to be susceptible to enteropeptidase action. Kinetic parameters of enteropeptidase hydrolysis for these substrates were determined.K _m values for all substrates with truncated linker (≈10⁻³ M) are an order of magnitude higher than corresponding values for typical enteropeptidase artificial peptide or fusion protein substrates with full enteropeptidase linker-DDDDK-(K _m ≈10⁻⁴ M).k _cat values for AT, Hb (2–8), WDDRG and WDDKG are ≈30–40 min⁻¹. But one additional amino acid residue at both N-and C-terminus of Hb (2–8) results in a drastic increase of hydrolysis efficiency:k _cat value for Hb (1–9) is 1510 min⁻¹. Recent study demonstrates the possibility of undesirable cleavage of target peptides or proteins containing the above-mentioned truncated linker sequences; further, the ability of enteropeptidase to hydrolyse specifically several biologically active peptidesin vitro along with its unique natural substrate trypsinogen was demonstrated.     

A carbon nanotube/silica sol–gel architecture for immobilization of horseradish peroxidase for electrochemical biosensor

A novel third-generation biosensor for hydrogen peroxide (H₂O₂) has been constructed based on horseradish peroxidase (HRP) immobilized by the sol–gel (SG) technology on carbon nanotube (CNT)-modified electrode. CNT has good promotion effects on the direct electron transfer between HRP and the electrode surface and the SG network provides a biocompatible microenvironment for enzyme. The immobilized HRP retained its bioelectrocatalytic activity for the reduction of hydrogen peroxide and can respond to the change of concentration of H₂O₂ rapidly. The heterogeneous electron transfer rate constant was evaluated to be 2.8 ± 0.4 s⁻¹. The amperometric response to H₂O₂ shows a linear relation in the range from 0.5 to 300 μmol l⁻¹ and a detection limit of 0.1 μmol l⁻¹ (S/N = 3). The K _M^app value of HRP immobilized on the electrode surface was found to be 1.35 mmol l⁻¹. The biosensor exhibited high sensitivity, rapid response and excellent long-term stability.     

Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D₂O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D₂O medium with protiated methanol as carbon source, or in 95% D₂O medium containing deuterated methanol. A high level of uniform C deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [¹H/¹⁵N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues, including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of ²H/¹³C/¹⁵N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.     

New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4' positions and non-hydrazine linkers

We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine bridge which joins the two palindromic moieties and the phenylalanine residues at the 4,4′ positions of the backbone. The new analogues 9 and 10 contain 1,2-phenylenediamine and piperazine, respectively, in place of the hydrazidic linker and p-fluoro-l-phenylalanine residues at 4 and 4′ positions. Binding values are: K_\texti^m = 0.51 \textnM K_{\text{i}}^{\mu } = 0.51\,{\text{nM}} and K_\texti^d = 12.8 \textnM K_{\text{i}}^{\delta } = 12.8\,{\text{nM}} for compound 9, K_\texti^m = 0.09 \textnM K_{\text{i}}^{\mu } = 0.09\,{\text{nM}} and K_\texti^d = 0.11 \textnM K_{\text{i}}^{\delta } = 0.11\,{\text{nM}} for analogue 10.