Fluorescent Gramicidin Derivatives for Single-Molecule Fluorescence

Tyler Lougheed, Vitali Borisenko, Christine E. Hand, and G. Andrew Woolley*. Department of Chemistry, 80 St. George Street, University of Toronto, Tor...
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Bioconjugate Chem. 2001, 12, 594−602

Fluorescent Gramicidin Derivatives for Single-Molecule Fluorescence and Ion Channel Measurements Tyler Lougheed, Vitali Borisenko, Christine E. Hand, and G. Andrew Woolley* Department of Chemistry, 80 St. George Street, University of Toronto, Toronto, Ontario M5S 3H6, Canada. Received January 22, 2001; Revised Manuscript Received April 12, 2001

Single-molecule spectroscopies in combination with single-channel patch-clamp measurements have the potential for providing new information on ion channel gating processes. Fluorescent gramicidin derivatives could provide a means for calibrating such experiments since the structure of the open channel is known and the mechanism of gating (peptide dimerization) is generally agreed. We describe here the synthesis and characterization of two pairs of gramicidin derivatives that should prove useful for such studies. They contain robust fluorophores, undergo resonance energy transfer (FRET) when they dimerize, and have single-channel properties close to those of the wild-type channel.

INTRODUCTION

Ion channels have been subjects of intensive study ever since the demonstration of their central importance for the function of nerve and muscle tissue (1, 2). The molecular events underlying channel gating (opening and closing) are of particular interest (3, 4). The difficulty of producing large quantities of these proteins and the necessity of maintaining a membrane environment for their proper function have hampered NMR-based and X-ray-based structural studies (5-7). Single-molecule fluorescence spectroscopy, in combination with site-specific fluorescent-labeling strategies and simultaneous electrical detection of ion flux, offers the potential for obtaining structural information on the gating process under near-native conditions (8, 9). The possibility of obtaining information on structural rearrangements accompanying gating using simultaneous fluorescence and electrical measurements under multichannel conditions has already been demonstrated by Isacoff and colleagues and by Cha and Bezanilla (1013). Single-molecule optical measurements offer the possibility of more detailed structural analyses. For instance, substates in opening transitions, that might be obscured in a multichannel analysis, could be resolved (14). The potential of combined optical and electrical detection for providing new information on the structures and dynamics of single ion channels has been realized (15), and several reports demonstrating the feasibility of such studies have appeared (16-19). In particular, singlemolecule fluorescence resonance energy transfer (FRET)1 could provide a direct means for probing structural changes underlying channel gating. Several reports of single-molecule fluorescence resonance energy transfer (FRET) have now appeared (20-24). To fully exploit the power of single-molecule optical methods to extract structural information on complex membrane proteins, it will be useful to have wellcharacterized model systems with which to calibrate the measurements (16). Changes in apparent FRET ef* To whom correspondence should be addressed at the Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada. E-mail: [email protected], Phone/Fax: (416) 978-0675.

ficiency can arise from several sources including reversible transitions to nonemitting states (‘blinking’) (25) and dye dynamics (26) (e.g., cis-trans isomerizations). Failure to account for different photostates can lead to errors in the interpretation of FRET experiments (27). The ion channel formed by the peptide gramicidin (HCO-Val-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-LeuTrp-D-Leu-Trp-D-Leu-Trp-NHCH2CH2OH) meets the requirements of a model system for simultaneous optical and electrical detection of ion channel gating. The structure and function of the gramicidin channel are wellestablished (28-30). Indeed, simultaneous fluorescence and conductance measurements with gramicidin (in a multichannel configuration) were reported 25 years ago by Veatch and Stryer (31). This classic work helped establish the dimeric model for the gramicidin channel. Gramicidin is hydrophobic and readily incorporates into bilayers when added to the aqueous phase nearby. Movement of gramicidin monomers in the plane of the membrane is relatively fast [diffusion rates measured using fluorescence recovery after photobleaching techniques are similar to those measured for fluorescently labeled lipids (32)]. The channel is formed upon association of two gramicidin monomers, one from each leaflet of the bilayer membrane, in an N-terminal to N-terminal (head-to-head) fashion (Figure 1) (28, 29, 33). The β6.3 helical structure of the peptide provides a pore ∼28 Å long and 4 Å in diameter that traverses the membrane when a dimer forms. The pore is lined by backbone amide groups and permits the transmembrane flux of small monovalent cations at maximum rates of 106-107 ions 1 Abbreviations: BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; BLM, bilayer lipid membrane; C:M:W, chloroform:methanol:water; Cy5-NHS, 1-[5-(N-succinimidyloxycarbonyl)-pent-1-yl]-2-[5-(3,3-dimethyl-1-ethyl-5-sulonato-indolin-2-ylidene)-1,3-pentadien-1-yl]-3,3-dimethyl-3H-indolium-5sulfonate; DMF, dimethylformamide; EDA, ethylenediamine; FRET, fluorescence resonance energy transfer; HPLC, highperformance liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MeOH, methanol; NHS, N-hydroxysuccinimidyl; PyMPO, benzyl-4-(5-(4-methoxyphenyl)oxazol-2yl)pyridinium; R6G, 5-(and 6)-carboxyrhodamine-6G; ROX, 5-carboxy-X-rhodamine; TEA, triethylamine; TFA, trifluoroacetic acid; TFE, trifluoroethanol; THF, tetrahydrofuran; TLC, thin-layer chromatography.

10.1021/bc010006t CCC: $20.00 © 2001 American Chemical Society Published on Web 06/01/2001

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Figure 1. Schematic diagram showing the dimerization of fluorescent gramicidin derivatives in a lipid bilayer. The bilayer is represented as two parallel lines. Dimerization provides a pathway for monovalent cations (small circles) through the membrane. This cation flux can be detected electrically at the single-channel level. Dimerization is also expected to permit efficient fluorescence resonance energy transfer (FRET) between donor (gram-R6G) and acceptor (gram-Cy5) peptides. In principle, this FRET signal could also be detected at the single-channel level.

per second. The channel is stabilized by six hydrogen bonds at the dimer junction. Channel closing is believed to be due to dissociation of the dimer into monomers. At 25 °C, the average lifetime of a dimer is on the order of 1 s (34, 35). If gramicidin dimerization could be detected optically at the single-molecule level, one would expect the optical change to coincide with a change in ionic current, measured electrically, through the membrane. With fluorescently labeled gramicidin derivatives, optical detection of peptide dimerization should be possible by measuring resonance energy transfer that would occur between dyes with appropriate absorption and emission spectra. Figure 1 shows the process diagrammatically. Peptides bearing a donor dye are added to one side of a lipid bilayer and peptides bearing an acceptor dye to the other. Transmembrane ionic current is measured using conventional patch-clamp or BLM techniques, and dimerization is detected as a step increase in this current. Upon peptide dimerization, the donor and acceptor dyes are 50 Å or less apart, a distance that should lead to efficient resonance energy transfer for many donor-acceptor pairs (36). We describe here the synthesis and characterization of two pairs of fluorescently labeled gramicidin derivatives that should prove useful for combined optical and electrical single-molecule studies. The synthetic methods are general and could be used to prepare other fluorescent gramicidin derivatives from commercially available amine-reactive dyes. We show that the derivatives incorporate into unilamellar lipid vesicles and into planar bilayers. Single-channel properties of the derivatives measured electrically in planar bilayers were found to be similar to those of the unmodified channel. When donor and acceptor peptides are incorporated into the same lipid vesicles, fluorescence resonance energy transfer was observed under conditions where the channel can be shown to be active using a diffusion potential assay.

Thus, the fluorescent gramicidin derivatives appear to have properties suitable for combined optical and electrical single-molecule experiments. Some further requirements for such experiments are considered. MATERIALS AND METHODS

1. Synthesis of Gramicidin-EDA (Gram-EDA). The C-terminal end of gramicidin was derivatized as described previously to install a primary amino group (37). Briefly, commercial gramicidin D (38 mg, 20 µmol) was combined with p-nitrophenyl chloroformate (200 µmol) in dry tetrahydrofuran (2 mL) (4 °C), and then triethylamine (TEA) (100 µL) was added and the mixture stirred for 1 h at 4 °C. The resulting carbonate ester was filtered through Celite into a 100-fold molar excess of ethylenediamine in 2 mL of dimethylformamide (DMF). The product [gramicidin-ethylenediamine (gram-EDA)] was then separated by gel filtration using Sephadex LH-20 (2.5 × 20 cm gravity column) in methanol. The product was further purified using reverse-phase HPLC [ZorbaxRX-C8 column (4.6 × 250 mm), isocratic conditions, 80% MeOH/20% H2O, 0.1% TFA adjusted to a pH of 3.0 with TEA, flow rate ) 1 mL/min, retention time 6.2 min]. Electrospray mass spectrometry: gram-EDA ) C102H147N22O18 (M+H+) calcd ) 1969.4, obsd ) 1969.5; TLC (C:M:W ) 65:25:4): gramicidin Rf ) 0.70, gram-EDA Rf ) 0.47. 2. Synthesis of Gramicidin-Rhodamine-6G (GramR6G). Gramicidin-EDA (2.0 mg, 1.02 µmol) was dissolved in 1.0 mL of dry DMF in a round-bottom flask under a nitrogen atmosphere. To the stirring solution of gramEDA was added 100 µL of dry TEA. In a separate reaction vessel, also under nitrogen, 1.0 mg (1.8 µmol) of (5/6)carboxyrhodamine-6G-NHS ester (Molecular Probes) was dissolved in 1.0 mL of dry DMF. The gram-EDA solution was then added dropwise over a period of 1 h via syringe to the stirring dye solution. After the reaction had stirred

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for 24 h, protected from light, the DMF was removed under high vacuum and the product resuspended in methanol and separated from unreacted dye using gelfiltration chromatography (LH-20 in methanol). The product was further purified using reverse-phase HPLC [Zorbax-RX-C8 column (4.6 × 250 mm), isocratic conditions, 80% MeOH/20% H2O, 0.1% TFA adjusted to a pH of 3.0 with TEA, flow rate ) 1 mL/min, retention time 11.6 min]. MALDI mass spectrometry: gram-R6G ) C129H171N24O22 (M+H+) calcd ) 2409.8, obsd ) 2409.7; TLC (C:M:W ) 65:25:4): gramicidin Rf ) 0.70, gram-R6G Rf ) 0.77. 3. Synthesis of Gramicidin-Cy5 (Gram-Cy5). Gramicidin-Cy5 was prepared in a manner similar to that described for gram-R6G. Gram-EDA (0.4 mg, 0.2 µmol) was mixed with 20 µL of dry TEA in 0.2 mL of dry DMF; then the contents of one vial (nominally 0.2-0.3 mg) of Cy5-NHS-ester (monoreactive)(Amersham Pharmacia Biotech) dissolved in 0.2 mL of dry DMF were added dropwise. This solution was stirred at room temperature overnight protected from light. The solvent was removed under high vacuum, and the residue was dissolved in 1 mL of methanol. The product was then purified by HPLC [Zorbax-C8 column (4.6 × 250 mm), isocratic conditions, 98% solvent A (0.1% TFA in 80% MeOH/20% H2O)/2% solvent B (0.1% TFA in H2O), flow rate ) 1 mL/min, retention time 5.4 min]. MALDI mass spectrometry (negative ion mode): gram-Cy5 ) C135H183N24O25S2 calcd ) 2606.2, obsd ) 2605; TLC (C:M:W ) 65:25:4): gramicidin Rf ) 0.70, gram-Cy5 Rf ) 0.44. 4. Synthesis of Gramicidin-PyMPO (GramPyMPO). PyMPO-NHS [1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2yl)pyridinium iodide] (Molecular Probes) (1.7 mg, 3 µmol) was combined with 0.17 mL of dry DMF, vortexed, and added slowly to 1 mL of dry DMF containing gram-EDA (1.2 mg, 1 µmol). After 1 h, dry TEA (20 µL, 0.14 mmol) was added, and the solution was stirred overnight at room temperature protected from light. The DMF was pumped off, and the product was purified by gel filtration using LH-20 in methanol. The product was further purified using reversephase HPLC [Zorbax-RX-C8 column (4.6 × 250 mm), isocratic conditions, 10% MeOH/90% solvent B (80% MeOH/20% H2O, 0.1% TFA adjusted to a pH of 3.0 with TEA), flow rate ) 1.2 mL/min, retention time 6.2 min]. MALDI mass spectrometry: gram-PyMPO ) C125H163N24O21 (M+) calcd ) 2337.7, obsd ) 2336.9; TLC (C:M:W ) 65:25:4): gramicidin Rf ) 0.70, gram-PyMPO Rf ) 0.76. 5. Synthesis of Gramicidin-ROX (Gram-ROX). ROX-NHS (5-carboxy-X-rhodamine-N-hydroxysuccinimidyl ester, Molecular Probes, 1.9 mg, 3 µmol) was reacted with gram-EDA using the same procedure as described for gram-PyMPO. After purification using the LH-20 column, the gram-ROX was further purified with a silica gel column (2 × 20 cm) using C:M:W ) 65:25:4 as eluant. The desired product was the first band eluted. The product was then purified using reverse-phase HPLC [Zorbax-RX-C8 column (4.6 × 250 mm), isocratic conditions, 15% MeOH/85% solvent B (80% MeOH/20% H2O, 0.1% TFA adjusted to a pH of 3.0 with TEA), flow rate ) 1.2 mL/min, retention time 10 min]. MALDI mass spectrometry: gram-ROX ) C135H175N24O22 (M+H+) calcd ) 2485.9, obsd ) 2485.5; TLC (C:M:W ) 65:25:4): gramicidin Rf ) 0.70, gram-ROX Rf ) 0.72. 6. Fluorescence Measurements of Gramicidin Derivatives in Lipid Vesicles. Unilamellar lipid vesicles containing gramicidin derivatives were prepared as follows: An aliquot of dioleoylphosphatidylcholine (DOPC)

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(Avanti Polar Lipids) (2 mg, 2.54 µmol) dissolved in methanol was added to a 1.5 mL Eppendorf tube containing either a given number of moles (e.g., 1 nmol) of an individual fluorescent gramicidin derivative or a 1:1 mixture (e.g., 0.5 nmol each) of two fluorescent gramicidin derivatives (total methanol volume ) 400 µL). Concentrations of fluorescent gramicidins were determined by UV-Vis absorbance measurements of stock solutions in methanol (extinction coefficients used: gram-R6G ) 92 000 at 520 nm, gram-Cy5 ) 250 000 at 649 nm, gramPyMPO ) 26 000 at 415 nm, gram-ROX ) 78 000 at 574 nm). Methanol was then removed under a gentle stream of dry nitrogen to form a thin lipid film, and the film was pumped under high vacuum for >2 h. The lipid/gramicidin film was resuspended in 400 µL of 400 mM KCl, 6.5 mM phosphate buffer, pH 7.0. Unilamellar vesicles containing fluorescent gramicidins were then formed by repeated extrusion (20 passes) of the lipid suspension through a polycarbonate membrane (Avanti Polar Lipids) with a pore diameter of 100 nm. Fluorescence measurements were made of vesicle solutions prepared from 1:1 mixtures of fluorescent gramicidins and compared to measurements of 1:1 mixtures of vesicle solutions prepared from pure fluorescent gramicidins. Final lipid concentrations and peptide concentrations are given in the figure legends. Steady-state fluorescence measurements were made using a Perkin-Elmer LS50B spectrofluorometer (equipped with a red-sensitive photomultiplier for Cy5 detection) and a 150 µL cuvette with 0.5 cm excitation and emission path lengths. All measurements were made at room temperature (23 ( 2 °C). 7. Fluorescence Detection of Diffusion Potentials Generated by Gramicidin in Lipid Vesicles. The establishment of a diffusion potential across lipid vesicle membranes was assayed using the potential-sensitive fluorescent dye safranine O (38). Unmodified gramicidin (0.1, 1, 3, 5, 50 nmol) was combined with lipid (32 µmol) in methanol. Vesicles were then prepared exactly as described above except that 500 µL of 200 mM KCl, 5 mM phosphate buffer, pH 7.0, was used. After extrusion through 100 nm polycarbonate filters, 20 µL of this vesicle solution was added to 3 mL of a solution of 340 mM sucrose, 5 mM phosphate buffer, pH 7.0, containing 1 µM safranine O (Aldrich). The fluorescence emission of safranine O was monitored at 581 nm (excitation 521 nm, 5 nm slits, 0.5 s response time). 8. Single-Channel Conductance Measurements of Gramicidin Derivatives. The general techniques for making single-channel conductance measurements of gramicidin derivatives have been described previously (39). A solution of 1 M CsCl, 5 mM BES, pH 7.0, was used as the electrolyte. Gramicidin derivatives were HPLC-purified at least twice (using the conditions given above) before use in single-channel measurements to ensure highly uniform conductance events (40). Peptides (∼10 nM in methanol) were added to membranes formed from diphytanoylphosphatidylcholine/decane (50 mg/mL)(Avanti Polar Lipids). Membranes were formed by painting the lipid solution across a ∼100 µm aperture in a polypropylene pipet tip mounted horizontally in a Teflon well. Silver/silver chloride electrodes were placed, one on either side of the membrane, and were connected to the CV-4B headstage of an Axopatch 1D patch-clamp amplifier (Axon Instruments) controlled by Synapse (Synergistic Research Systems) software that permitted transmembrane voltage to be set and current recorded using the same pair of electrodes. Current records were filtered

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Figure 2. Chemical structures of gramicidin derivatives.

at 50 or 100 Hz, sampled at 1 kHz, stored directly to disk, and analyzed using Synapse and Igor (Wavemetrics Inc.) software. RESULTS AND DISCUSSION

Synthesis of Fluorescently Labeled Peptides. Gramicidin isolated from Bacillus brevis is a mixture of peptides (denoted gramicidin D) with the general structure formyl-Xxx-Gly-Ala-D-Leu-Ala-D-Val-Val-D-Val-TrpD-Leu-Yyy-D-Leu-Trp-D-Leu-Trp-NHCH2CH2OH, where Xxx is Val or Ile and Yyy is Trp (gramicidin A), Phe (gramicidin B), or Tyr (gramicidin C) (Figure 2) (41). Previous work with fluorescent gramicidins (31, 32, 4244) relied primarily on a dansylated (Tyr11-O-dansyl) gramicidin C derivative. Although this derivative was reported to have channel properties similar to those of native gramicidin, modifications at position 11 in the sequence to install various fluorescent groups might not be generally well tolerated (28). We decided instead to focus on derivatization of the C-terminal end of the peptide since chemical modifications at this end (the membrane/solution interface) do not significantly affect the overall structure of the channel (29). Chemical modifications were carried out on the mixture (gramicidin D) since it is predominantly (>80%) Val1-gramicidin A and, in any case, all peptides are HPLC-purified after modification, which removes minor species. Val1-gramicidin A has no functional groups suitable for reaction with typical fluorescent labeling reagents, so we first derivatized the C-terminal end to provide a convenient site for attachment of commercially available amine-reactive fluorescent labels (37). To be useful for single-molecule work, fluorescent dyes must be robust (i.e., resistant to photobleaching). We therefore focused on cyanine-based and rhodamine-based dyes that have been used successfully for these purposes (45). In addition, for fluorescence resonance energy transfer (FRET) measurements, dyes with overlapping emission and excitation spectra are required (46). The amine-functionalized gramicidin (gram-EDA, Figure 2) was reacted with four different fluorescent labels (rhodamine-6G, Cy5, PyMPO, ROX) all purchased as N-hydroxysuccinimidyl esters. Each product was purified by gel filtration chromatography in methanol (the pep-

tides are not water soluble), followed by reverse-phase high-performance liquid chromatography (HPLC) to ensure that no unreacted gram-EDA or unreacted dye was present (40). Mass spectrometry [matrix-assisted laser desorption ionization (MALDI)], in addition to absorbance and fluorescence data, confirmed the identity of the products. The chemical structures of the four fluorescently labeled gramicidin derivatives are shown in Figure 2. In addition, uniform single-channel conductance properties (vide infra) are an excellent indication of purity (40). Channel Formation in Lipid Vesicles. Before performing fluorescence measurements, we wished to ensure that the fluorescent gramicidins were incorporated into a membrane in a functional form. Accordingly, dilute solutions of the peptides were combined with lipid in methanol. Under these conditions, the peptides are expected to be predominantly monomeric (47). As the methanol is evaporated, the peptide forms an intimate mixture with the lipid. When aqueous buffer is then added and the lipid/peptide suspension is extruded through 100 nm polycarbonate filters, unilamellar lipid vesicles are expected to form with peptide incorporated in the single-helix (channel-forming) structure. Although gramicidin peptides are known to form intertwined double helices in organic solvents, these convert to the single-helix form in the presence of lipid (47). Polar organic solutions facilitate this conversion. In particular, trifluoroethanol has been reported to function well in this regard (48). However, we found that traces of TFE remaining after extensive drying led to interference with fluorescence measurements, particularly where membrane potential was assayed using safranine O (see below). Consequently, we used methanol as the solvent for mixing peptide and lipid. Lipid vesicles of 100 nm diameter may be estimated to contain ∼100 000 molecules of lipid taking an average area per lipid molecule of 0.6 nm2 (49). If one takes 1 nmol of gramicidin and 2.5 µmol of lipid, an average of 40 gramicidin molecules per vesicle is obtained. This corresponds to a total surface density of gramicidin in the membrane of 2.3 × 10-13 mol/cm2.2 The fraction of dimeric (active channel) form will depend on the dimerization constant for the particular membrane type.

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Dimerization constants have been estimated for the dansyl gramicidin C derivative in dioleoylphosphatidylcholine (DOPC)/decane membranes by Veatch et al. (31) to lie in the range of 1013-1014 cm2/mol. Values between 1014 (for 0 mV) and 1015 cm2/mol (for 200 mV) have been reported for gramicidin A by Bamberg and La¨uger (35) based on current relaxation measurements also in DOPC/ decane membranes (the voltage dependence arises from a voltage-induced thinning of the membrane). If we use the same range of values for the present case, 63-95% of the peptide is estimated to be in dimeric form. As the concentration of peptide is lowered, the equilibrium fraction of dimers decreases; however, even with only two gramicidin molecules per vesicle, ion transport should still be possible over time. Transport should not be possible in vesicles with less than two peptide molecules since one molecule cannot form a channel and molecules cannot move between vesicles assuming there is no vesicle fusion (50). To test the prediction that active channels should be present in vesicles when two or more peptide molecules are present, we investigated whether a diffusion potential could be established across the vesicle membranes. Vesicles were prepared in 200 mM KCl solution and diluted into isotonic sucrose solution. Were a gramicidin channel present, K+ ions should diffuse out of the vesicle, but, since no other cations are present and gramicidin is impermeable to anions, a potential should develop, negative inside, that opposes K+ efflux. Development of this potential can be followed by observing the redistribution of the cationic fluorescent dye safranine O between aqueous and membrane-bound compartments (38). Figure 3 shows the fluorescence response observed with various concentrations of (unmodified) gramicidin per vesicle. The channel is clearly active. As expected, no fluorescence response is seen if equimolar NaCl is included in the external medium (data not shown), in keeping with the known ability of gramicidin to transport sodium ions. Fluorescence Spectroscopy. Having established that the present procedure for incorporating gramicidin channels into lipid vesicles led to active channel species, we wished to examine the fluorescence properties of these channels and to test whether the fluorescent tags would serve as functional donor-acceptor pairs for use in fluorescence resonance energy transfer studies. Figure 4 shows normalized fluorescence excitation and emission spectra for each of the four fluorescent gramicidins incorporated (individually) into lipid vesicles. The small size of the lipid vesicles used (100 nm diameter) results in light scattering intensities at these wavelengths that can be readily separated from fluorescence (51). To test for resonance energy transfer, two different types of sample containing vesicle-bound fluorescent gramicidins were prepared. In case A, a vesicle solution containing only donor peptide and a vesicle solution containing only acceptor peptide were prepared, and then these vesicle solutions were mixed in a 1:1 ratio. In case B, donor and acceptor peptides were mixed directly in a 1:1 ratio and then incorporated together into vesicles. The result is two solutions with equal concentrations of lipid, donor peptide, and acceptor peptide, but with different distributions of the donors and acceptors. Since the aqueous solubility of the peptides is very low, the peptides are not expected to redistribute between vesicles (50). In case A, the following molecular species may be present: 2 Forty molecules of peptide (6.6 × 10-23 mol) divided by the area of a 100 nm diameter vesicle (2.9 × 10-10 cm2).

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Figure 3. Fluorescence detection of channel formation in vesicles. At the time indicated by the arrow, K+-loaded vesicles alone (0) or K+-loaded vesicles containing gramicidin (average number of gramicidin molecules per vesicle is indicated) were added to K+-free buffer containing isotonic sucrose and the potential-sensitive dye safranine O. Concentrations are given under Materials and Methods. Fluoresence enhancement is observed as the dye associates with vesicle membranes in response to the development of a K+ diffusion potential (38).

Figure 4. (A) Excitation and emission spectra (normalized) of gram-R6G and gram-Cy5 in lipid vesicles: (- ‚ -) gram-R6G excitation, (‚‚‚) gram-R6G emission, (s) gram-Cy5 excitation, (- - -) gram-Cy5 emission. (B) Excitation and emission spectra (normalized) of gram-PyMPO and gram-ROX in lipid vesicles: (- ‚ -) gram-PyMPO excitation, (‚‚‚) gram-PyMPO emission, (s) gram-ROX excitation, (- - -) gram-ROX emission.

donor monomers, donor dimers, acceptor monomers, and acceptor dimers. In the premixed sample (case B), donoracceptor dimers (i.e., FRET pairs) will also be present. Since the concentration of vesicles in these experiments is ∼64 nM (6.4 mM DOPC present in vesicles of 100 000

Fluorescent Gramicidin Derivatives

Figure 5. (A) Fluorescence resonance energy transfer between gram-R6G and gram-Cy5: (s) gram-R6G (0.3 µM) and gramCy5 (0.31 µM) in separate vesicles (6.4 mM DOPC); (- - -) gramR6G (0.31 µM) and gram-Cy5 (0.34 µM) in the same vesicles (6.4 mM DOPC), excitation at 490 nm, 10 nm slit widths. (B) Fluorescence resonance energy transfer between gram-PyMPO and gram-ROX: (s) gram-PyMPO (1.1 µM) and gram-ROX (0.9 µM) in separate vesicles (6.4 mM DOPC); (- - -) gram-PyMPO (1.3 µM) and gram-ROX (1 µM) in the same vesicles (6.4 mM DOPC), excitation at 422 nm, 15 nm slit widths.

molecules each), the average separation between vesicles is ∼3000 Å. This value is much larger than the expected Fo¨rster distance (∼50 Å) so that FRET should only be possible in case B, where donor and acceptor gramicidins are co-incorporated into the same vesicles. Figure 5A compares the fluorescence spectra obtained in case A and case B for the gram-R6G/gram-Cy5 pair where the peptide:lipid ratio is ∼1:10 000. Figure 5B compares the spectra obtained with the gram-PyMPO/ gram-ROX pair where the peptide:lipid ratio is ∼1:3200. In each case, significant quenching of donor fluorescence is observed for case B, the premixed samples, in comparison to case A. Additional evidence for FRET is the enhancement of acceptor fluorescence in case B for each dye pair. Note that there is minimal direct excitation of the acceptor in the gram-R6G/gram-Cy5 case as compared to the gram-PyMPO/gram-ROX case. Energy transfer efficiencies were calculated by the method of Clegg et al. (52) using both donor quenching and acceptor enhancement data. Calculated transfer efficiencies were ∼23 ( 5% for the gram-R6G/gram-Cy5 pair and 60 ( 10% for the gram-PyMPO/gram-ROX pair for the particular conditions described in the legend to Figure 5. The lower transfer efficiency of the gram-R6G/gram-Cy5 pair is

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consistent with a lower peptide-to-lipid ratio, and therefore a smaller fraction of dimers, for that case. Increasing the peptide-to-lipid ratio gave increasing FRET efficiencies as expected; however, with acceptor concentrations >1 µM, self-quenching of Cy5 was observed (data not shown). In principle, FRET can also occur between donors and acceptors that are present in the same vesicle but not part of a donor/acceptor dimer. Such lateral energy transfer in lipid bilayers has been analyzed previously by several authors (53-56). While lateral FRET may be observed even when the average donor-acceptor distance exceeds the Fo¨rster distance severalfold (53), FRET will nevertheless occur most efficiently between donors and acceptors that are actually associated (55). The total surface density of gramicidin in the membrane in the present FRET experiments ranges from 5.8 × 10-14 to 2.3 × 10-13 mol/cm2, which corresponds to average distances between noninteracting peptides in the range of 550-270 Å if they were monomeric and 750-380 Å if they were dimeric. These values are larger than the expected Fo¨rster distances for these dye pairs, and substantially larger than the distance between donor and acceptor dyes in a gramicidin dimer ()50 Å). Since the fluorescent gramicidin peptides are able to form active dimers as demonstrated by the conductance data in bilayers (vide infra), we expect the observed FRET to be due primarily to the presence of heterodimer (donoracceptor) gramicidin channels rather than the mere coexistence of the donor and acceptor peptides in the same vesicles. The observed energy transfer efficiency is affected not only by the ratio of dimers to monomers and the intrinsic efficiency for the particular dye pair as it occurs in the gramicidin dimer, but also by the fact that donors can be paired with donors as well as with acceptors. If we assume that the dimerization constants for all possible dimers [donor-donor, acceptor-donor (two orientations), acceptor-acceptor] are the same, then approximately half of all dimers present should be FRET pairs since the concentrations of donors and acceptors are approximately equal. Single-molecule FRET measurements with these systems would thus be expected to show approximately double the energy transfer efficiency determined from a macroscopic measurement on dimerized gramicidins. Channel Formation in Black Lipid Membranes. The single-channel properties of each of the fluorescent gramicidin derivatives were examined in planar lipid bilayers using 1 M CsCl as the electrolyte. Single-channel current recordings are shown in Figure 6. In general, the single-channel properties of all the derivatives are similar to native gramicidin channels measured under the same conditions (see Table 1) (57). This result is expected as the C-terminal modifications are not expected to affect channel function significantly (29). A distinguishing feature of the modified channels, however, is the small steplike current fluctuations superimposed on the main conducting level in each case (Figure 6). Steps in current recording are also observed with gramicidin-EDA of the same average duration (∼50 ms) and are due to thermal cis-trans isomerization of the carbamate group that is introduced near the channel mouth that alters the position of the protonated amino group relative to the channel mouth (58). The size of the current steps is much smaller for the fluorescent derivatives than for gramEDA because charges on the fluorescent groups are significantly further from the channel entrance [cf. (59)]. The variation of the step sizes between the different fluorescent derivatives is also expected since isomeriza-

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Table 1. Single-Channel Properties of Fluorescent Gramicidin Derivatives

mean lifetimea (s) conductanceb (pS)

gram A (n ) 250)

gram-R6G (n ) 96)

gram-Cy5 (n ) 91)

gram-PyMPO (n ) 23)

gram-ROX (n ) 129)

0.4 46

4 45

2.3 45

0.8 45

1.8 43

a Estimated as described in (63). b Measured at 200 mV, 1 M CsCl, using diphytanoylphosphatidylcholine/decane membranes. Errors are on the order 5%.

Figure 6. Representative single-channel current recordings of (a) gram-R6G, (b) gram-Cy5, (c) gram-PyMPO, and (d) gram-ROX in diphytanoylphosphatidylcholine membranes. The applied voltage was 200 mV, and 1 M CsCl was used as the electrolyte.

tion of the linkage connecting the fluorescent group to the rest of the peptide is likely to have different effects on ion access in each case. These small fluctuations may be considered signatures of the modified channels that distinguish them from possible gram-EDA or native gramicidin contaminants in the membrane. Isomerization might also be expected to affect FRET efficiency in timeresolved measurements on single molecules, and this feature of the modified channels might also provide an internal calibration for single-molecule optical measurements. Toward Simultaneous Single-Molecule Optical and Electrical Measurements. Single-molecule electrical measurements with gramicidin and derivatives are straightforward and highly reproducible. The dye conjugates, with the exception of PyMPO, described here have been used successfully in single-molecule optical detection experiments previously, and we have demonstrated that the two pairs can undergo efficient FRET when channels form. Correlating a single-molecule FRET measurement with a channel opening event measured electrically will require that single active channels (dimeric channels) can be observed among nonconducting monomers in a planar bilayer. Single-channel measurements with gramicidin are typically performed at peptide-to-lipid ratios