Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.     

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.     

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.     

Investigation of biotin-streptavidin binding interactions using microcantilever sensors

We report the investigation of biotin-streptavidin binding interactions using microcantilever sensors. A symmetric cantilever construction is employed to minimize the effects of thermal drift and the control of surface chemistry on the backside of the cantilever is demonstrated to reduce the effects of non-specific binding interactions on the cantilever. Three structurally different biotin modified cantilever surfaces are used as a model system to study the binding interaction with streptavidin. The cantilever response to the binding of streptavidin on these biotin sensing monolayers is compared. The lowest detection limit of streptavidin using biotin-HPDP is found to be between 1 and 10nM limited by the optical measurement setup. Surface characterization using quartz crystal microbalance (QCM) and high-resolution atomic force microscope (AFM) is used to benchmark the cantilever sensor response. In addition, the QCM and AFM studies reveal that the surface density of bound streptavidin on biotin modified surfaces was low, thereby implying that effects other than steric hindrance are responsible for defining cantilever response.     

Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea

We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.     

The sensor regions of VDAC are translocated from within the membrane to the surface during the gating processes.

The motion of the sensor regions in a mitochondrial voltage-gated channel called VDAC were probed by attaching biotin at specific locations and determining its ability to bind to added streptavidin. Site-directed mutagenesis was used to introduce single cysteine residues into Neurospora crassa VDAC (naturally lacks cysteine). These were chemically biotinylated and reconstituted into planar phospholipid membranes. In the 19 sites examined, only two types of results were observed upon streptavidin addition: in type 1, channel conductance was reduced, but voltage gating could proceed; in type 2, channels were locked in a closed state. The result at type 1 sites is interpreted as streptavidin binding to sites in static regions close to the channel opening. The binding sterically interferes with ion flow. The result at type 2 sites indicates that these are located on a mobile domain and coincide with the previously identified sensor regions. The findings are consistent with closure resulting from the movement of a domain from within the transmembrane regions to the membrane surface. No single site was accessible to streptavidin from both membrane surfaces, indicating that the motion is limited. From the streptavidin-induced reduction in conductance at type 1 sites, structural information was obtained about the location of these sites.     

Calcium-dependent association of annexins with lipid bilayers modifies gramicidin A channel parameters

In order to examine whether calcium-dependent binding of annexin to acidic phospholipids could change the lipid bilayer environment sufficiently to perturb channel-mediated transmembrane ion-transport, gramicidin A channel activity in planar lipid bilayers was investigated in the presence of calcium and annexins II, III or V. The experiments were performed with membranes consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in 300 mM KCl solution buffered to pH 7.4 and with either 0.1 or 1 mM calcium added to the solution. Annexin (1 microM) was subsequently applied to the cis side of the membrane. All three annexins (II, III and V) when tested at 1 mM calcium decreased the gramicidin single-channel conductance. Annexins II and III increased the mean lifetime of the channels whereas annexin V seemed to have no influence on the mean lifetime. Since the lifetime of gramicidin A channels is a function of the rate constant for dissociation of the gramicidin dimer, which is dependent on the physical properties of the lipid phase, binding of annexins II and III seems to stabilize the gramicidin channel owing to a change of the bilayer structure.     

A streptavidin-biotin binding system that minimizes blocking by endogenous biotin

Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.     

Protein engineering of streptavidin for in vivo assembly of streptavidin beads

Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.     

Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers.     

A single-channel sensor based on gramicidin controlled by molecular recognition at bilayer lipid membranes containing receptor

A novel ion-channel sensor based on a membrane bound receptor and a single gramicidin channel is described, in which the binding of an analyte to the membrane bound receptor modulates the single-channel activity of gramicidin. The sensor is composed of a planar bilayer lipid membrane (BLM) containing biotin-labeled phosphatidylethanolamine as receptor for avidin and gramicidin as signal transducer. When the receptor catches an analyte (avidin or ferritin-labeled avidin (FA)) at the membrane surface, the bilayer structure is locally distorted and the gramicidin monomer/dimer kinetics is modulated in a manner that the fraction of channel opening with a short lifetime ( < or = 100 ms) to the total opening events increases. The fraction was found to increase with the concentration of avidin from 1.0 x 10(-9) to 1.0 x 10(-6) M and of FA from 1.0 x 10(-9) to 1.0 x 10(-8) M. With dinitrophenyl-labeled PE embedded as receptor in the BLM for monoclonal anti-dinitrophenyl antibody (anti-DNP), the fraction of channel openings ( < or = 100 ms) increased with the concentration of anti-DNP from 2.0 x 10(-9) to 2.0 x 10(-7) g/ml. Bovine serum albumin (BSA) and anti-BSA antibody caused no changes in the channel opening. The possible mechanism of analyte-induced modulation of single-channel activity of gramicidin is also discussed.     

Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.     

The dimeric nature of the gramicidin A transmembrane channel: Conductance and fluorescence energy transfer studies of hybrid channels

Gramicidin A, a linear peptide antibiotic, makes membranes permeable to alkali cations and hydrogen ions by forming transmembrane channels. We report here conductance and fluorescence energy transfer studies of channels containing two kinds of gramicidin. These studies of hybrid channels were designed to determine the number of molecules in a channel. The gramicidins studied were gramicidin A, dansyl gramicidin C, the p-phenylazobenzene sulfonyl derivative of gramicidin C (PABS⁴ gramicidin C), and the 4-(diethylamino)-phenylazobenzene-4-sulfonyl chloride derivative of gramicidin C (DPBS gramicidin C). The dansyl, PABS and DPBS groups were linked to the hydroxyl group of tyrosine 11 in gramicidin C. The single-channel conductance of PABS gramicidin C in planar bilayer membranes is 0.68 that of gramicidin A. Membranes containing both PABS gramicidin C and gramicidin A exhibit three kinds of channels: a pure gramicidin A, a pure PABS gramicidin C channel, and a hybrid channel with an intermediate conductance (0.82 that of gramicidin A). The dependence of the frequencies of these three kinds of channels on the mole fractions of gramicidin A and PABS gramicidin C in the membrane-forming solution fits a dimer model. Fluorescence energy transfer was used as a complementary means of ascertaining the frequency of hybrid channels. Dansyl gramicidin C was the fluorescent energy donor and DPBS gramicidin C was the energy acceptor. The efficiency of energy transfer between these chromophores in hybrid channels in liposomes was 75%. The relative quantum yield of the dansyl fluorescence was measured as a function of the mole fraction of DPBS gramicidin C. These fluorescence studies, like the single-channel conductance measurements, showed that there are two molecules of gramicidin in a channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes.     

Stable,high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (K_d = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (T_m = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc.     

Synthesis, characterization, and black lipid membrane studies of [7-L-alanine] gramicidin A

With a view to study the relevance of side-chain orientation in the transport of cations through a gramicidin transmembrane channel and to identify an analogue with favorable characteristics, [L-Ala7] gramicidin A was synthesized, purified, verified, and characterized by high-performance liquid chromatography, by carbon-13 and proton magnetic resonance spectra, and by circular dichroism spectra in methanol. Complete incorporation as the channel state was achieved when packaged in lysolecithin-containing lipid bilayers. The single-channel conductance data in diphytanoyllecithin/n-decane membranes are presented along with those of synthetic gramicidin A (GA). [L-Ala7] GA exhibits the highest most probable single-channel conductance so far reported for an analogue occurring at 28 pS as compared to 21 pS for GA under similar conditions. Also, a dramatic reduction in the dispersity of conducting states is observed with about 76% of the events falling in a narrow 1.75-pS conductance window as compared to about 31% of the events for GA under identical conditions. Thus, with the above characteristics, [L-Ala7]GA appears to be a very good candidate for a thorough study of ionic mechanism. The present results indicate that elements intrinsic to the channel proper are rate-limiting for GA and that there is no interfacial polarization or diffusion-controlled association at 1 M KCl and a 100-mV applied potential.     

Engineered streptavidin monomer and dimer with improved stability and function

Although streptavidin's high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.     

Detection of protein-ligand interaction on the membranes using C-terminus biotin-tagged alamethicin

C-terminal biotin-tagged alamethicin, which has several alpha-aminoisobutyric acid (Aib) residues in its sequence, was synthesized by the preparation of the protected peptide segment using the 2-chlorotrityl resin, followed by conjugation with biotin hydrazide. Suppression of the channel current of the biotin-tagged alamethicin by the addition of streptavidin to the electrolyte was monitorable in real time using the planar lipid-bilayer method. The system was also applicable to the detection of interaction of the biotin-tagged alamethicin with the anti-biotin antibody.     

Artifactual detection of biotin on histones by streptavidin

Biotinylation is a recent addition to the list of reported posttranslational modifications made to histones. Holocarboxylase synthetase (HCS) and biotinidase have been implicated as biotinylating enzymes. However, the details of the mechanism and the regulation of biotin transfer on and off histones remains unclear. Here we report that in a cell culture system low biotin availability reduces biotinylation of carboxylases, yet apparent biotinylation of histones is unaffected. This is despite biotin depletion having detrimental effects on cell viability and proliferation. Further analysis of the widely used method for detecting biotin on histones, streptavidin blotting, revealed that streptavidin interacts with histones independently of biotin binding. Preincubation of streptavidin with free biotin reduced binding to biotinylated carboxylases but did not block binding to histones. To investigate biotinylation of histones using an alternative detection method independent of streptavidin, incorporation of 14C biotin into biotinylated proteins was analyzed. Radiolabeled biotin was readily detectable on carboxylases but not on histones, implying very low levels of biotin in the nucleus attached to histone proteins (< 0.03% biotinylation). In conclusion, we would caution against the use of streptavidin for investigating histone biotinylation.     

The structure of the gramicidin A transmembrane channel

Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11. Simultaneous conductance and fluorescence measurements on planar lipid bi-layer membranes containing dansyl gramicidin C yielded four conclusions: (1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 ± 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. (2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel. (3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 Å-hydrocarbon thickness than in a 47 Å-glycerolester membrane. The dimerization constant in a 48 Å-phosphatidyl choline membrane was 200 times larger than in a 47 Å-glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. (4) We were readily able to detect 10^?14 mole cm^?2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square μm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes. An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazo-benzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a² and d² were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a tetramer channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. The various models which have been proposed for the conformation of the gramicidin transmembrane channel are briefly discussed.     

Shortened analog of the gramicidin A channel argues for the doubly occupied channel as the dominant conducting state

A shortened analog of the gramicidin A transmembrane channel has been synthesized and its transport characterized in planar lipid bilayer membranes. General considerations of a shorter diffusional length and a shorter distance over which the voltage drop occurs (i.e., an increased electric field) would contribute to an increase in single-channel conductance. The finding of a decreased single-channel conductance supports the perspective that the dominant conducting state is the doubly occupied channel wherein distance-dependent repulsion due to the first ion in the channel impedes entry of the second ion in the shorter channel.