4238
Langmuir 1998, 14, 4238-4242
Optical Modulation of the Insertion of Gramicidin into Bilayer Lipid Membranes Peter Osman,* Scott Martin, Dusan Milojevic, and Charles Tansey Collaborative Research Centre for Molecular Engineering & Technology, 126 Greville St., Chatswood, NSW 2067, Australia
Frances Separovic School of Chemistry University of Melbourne, Parkville, Vic 3052, Australia Received February 4, 1998. In Final Form: May 6, 1998 Gramicidin A (gA) has been synthesized with a photoisomerizable azobenzene group linked to the C-terminus of the channel. Using single-channel BLM measurements, we have demonstrated the effect of irradiating an optically gateable gramicidin on gramicidin channel conduction. A parallel study was carried out using UV and NMR spectroscopy of samples after dark adaptation, after exposure to UV between 330 and 340 nm, and after exposure to white light. A “photostationary” state of 3:1 trans-cis was observed on exposure to white light. It was found that channel formation was substantially increased after exposure to UV irradiation and that this was reversed after dark adaptation. These results were consistent with a mechanism in which membrane insertion of azobenzene-linked gA was optically modulated.
Introduction Gramicidin A (gA) is a 15 amino acid polypeptide, which dimerizes to form ion channels in bilayer lipid membranes (BLM). These channels have been well-defined in both structure and function.1-3 It has been known for many years that UV light can irreversibly alter the conduction of gA by cleaving the tryptophans.4 More recently reversible optical control of ion transport in covalently linked gramicidin monomers has been demonstrated.5,6 Stankovic et al.5 achieved this by linking the two gA monomers with a 3,3′-azobis(benzeneacetic acid) at the N- to N-terminus, which resides in the middle of the membrane bilayer. In the dark adapted state the transisomer forms channels very similar to those normally observed in unmodified gA. The irradiation induced a flickering change in conductivity superimposed on the normal conductivity signal of the dark-adapted trans isomer. This could be interpreted as photomodulation of the gA channel dimerization. Many gramicidin analogues have been synthesized with modifications at the C-terminus.7-11 Most of these have been shown to retain the characteristic square wave signal associated with gA. At most, reductions on the order of 20% can be seen in the single channel currents of such (1) Arseniev, A.; Barsukov, I. L.; Bystrov, V. F.; Lomize, A. L.; Ovchinnikov, Y. A. FEBS Lett. 1985, 186, 168-174. (2) Nicholson, L. K.; Cross, T. A.; Biochemistry 1989, 28, 9379-9385. (3) Koeppe, R. E., II.; Andersen, O. S. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 231-258. (4) Busath, D. D.; Waldbillig, R. C. Biochim. Biophys. Acta 1983, 736, 28-38. (5) Stankovic, C. J.; Heinemann, S. H.; Schreiber, S. L. Biochim. Biophys. Acta 1991, 1061, 163-170. (6) Lien, L.; Jaikaran, D. C. J.; Zhang, Z.; Woolley, A. J. Am. Chem. Soc. 1996, 118, 12222-12223. (7) Vogt, T. C. B, Killian, J. A.; de Kruijff, B. Biochim. Biophys. Acta 1991, 1069, 157-164. (8) Vogt, T. C. B.; Killian, J. A.; de Kruijff, B.; Andersen, O. S. Biochemistry 1992, 31, 7320-7324. (9) Seoh, S.A.; Busath, D. A. Biophys. J. 1993, 65, 1817-1827. (10) Woolley, G. A.; Jaikaran, A. S. I.; Zhuang, Z.; Peng, S. J. Am. Chem. Soc. 1995, 117, 448-454. (11) Cornell, B. A.; Braach-Maksvytis, V. B.; King, L.; Osman, P.; Raguse, B.; Wieczorek, L.; Pace, R. Nature 1997, 387, 580-583.
Figure 1. Preparation of the photoswitchable gramicidin derivatives via coupling of the known azobenzene derivatives (derived from the corresponding amines through MnO2 mediated oxidative dimerization) with gramicidin.
analogues. In general, modifications of the C-terminus have been shown to have a somewhat lesser effect on the gA ion channels than N-terminal modifications although Vogt, et al.8 have demonstrated that acylated gA is less effective in forming channels than native gA. Lien et al.6 have attached para- and meta-substituted azobenzene groups to the C-terminus of gA via a carbamate linkage and demonstrated photogating of the gA channel as manifested by increases in the repetition rate and amplitude of channels following irradiation with 337 nm light. The carbamate linkage undergoes thermal cis/trans isomerization12 whereas the azobenzene group undergoes photoisomerization from the trans to cis form with UV light. Lien et al.6 have demonstrated that the photomodulated blocking of the modified gA channels is reversible. In the present study, a photoswitchable gA derivative was prepared via coupling of azobenzene dicarboxylic acid directly to the ethanolamine at the C-terminus of gA (azogA) (Figure 1). Hence our azo-gA derivative possessed only one isomerizable bond and was terminated in a COOH group rather than an NH3+ group as in the case described (12) Jaikaran, D.C. J.; Woolley, G. A. J. Phys. Chem. 1995, 99, 1335213355.
S0743-7463(98)00135-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/01/1998
Optical Modulation of the Insertion of Gramicidin
Langmuir, Vol. 14, No. 15, 1998 4239
by Lien et al.6 The carboxylic acid group was used so as to be less disruptive to the channel function when compared with the protonated amine group, which was designed by Lien et al as a channel blocker of gA, which acts as a monovalent cation channel. The photoactive azobenzene group is in the trans-form in the dark-adapted state and in the cis-form following UV illumination. Photoisomerization of the azobenzene group attached to gA was expected to produce changes in ion channel properties, observable using electrophysiological techniques such as single-channel measurements of BLMs containing azo-gA. UV and NMR (nuclear magnetic resonance) spectroscopy also monitored light-induced changes in the structure of azo-gA in solution. The results were consistent with photomodulation of channel formation, with the cis form of azo-gA able to better insert into the membrane and form dimers. The paper investigates the effect of ultraviolet light on the insertion of photoactive gramicidin channels into lipid membranes. It seeks to discriminate between the optical opening and closing of the ion channel and modulation of the insertion of gramicidin into lipid bilayer membranes. Modified gA channels are being used in sensing devices fashioned from single lipid bilayers tethered to gold substrates.11 Materials and Methods The Synthesis of Azo-gA. The azobenzene unit was prepared by methods reported elsewhere in the literature.5,13 The synthesis of azo-gA is described in Appendix 1. The synthesized compounds were characterized by mass spectroscopy and UV and NMR spectroscopy. Light Sources. For UV (ultraviolet) irradiation, a 50 W Hg lamp with a 330-340 band-pass filter was used. Irradiations were carried out for a range of intervals depending on the sample. For dilute concentrations (nanomolar) as used for UV spectroscopy and single channel measurements, 0.5-1.5 h illumination was sufficient using filtered light from the Hg lamp. However, at higher concentrations of azo-gA (5 mM and 5 µM) as used for NMR, periods up to 7 h in UV light were required to achieve full conversion from the trans to the cis states. To revert to the all-trans form, the dark-adapted state, it was sufficient to place the azo-gA at 333 K for an hour. This was far more efficient than illumination using a Hg lamp with a 330-340 nm filter for periods of 90 min or longer. To achieve the photostationary state, samples were exposed to bright sunlight for approximately 1 h. This resulted in a trans-to-cis ratio of approximately 3:1. UV and NMR Spectroscopic Assays. UV spectra in the range 300-525 nm of solutions in dimethyl sulfoxide (DMSO) were recorded in 1 mL quartz cuvettes (1 cm path length) on a UV-visible spectrophotometer (CAREY 5, Varian, Palo Alto, CA). Proton NMR spectra were obtained using a 400 MHz spectrometer (UNITY INOVA, Varian, Palo Alto, CA) with typically a 5000 Hz sweep width and 1 Hz line broadening. Samples were recorded in DMSO-d6 using DMSO-d5-H (m ) 2.49 ppm) as an internal standard. Spectra were also obtained in deuterated methanol for structural comparison with native gA. Single-Channel Recordings. Stable bilayer lipid membranes were formed by painting a 100 mg mL-1 solution of freshly prepared glycerol-monooleate (Avanti, Alabaster, AL) in tetradecane (Sigma, St. Louis, MO) across a 100 µm hole formed in a Perspex septum. Experiments were carried out in symmetrical unbuffered 1 M NaCl solutions prepared with filtered deionized water (Millipore, Bedford, MA). The gramicidin solutions were prepared in ethanol and stored at 273 K and allowed to equilibrate at room temperature prior to injection into a BLM chamber. Once a stable membrane was formed, between 5 and 20 µL of 10 nM gramicidin solution was injected into both compartments of the chamber, which had a total volume of 7 mL. (13) Wheeler, O. H.; Gonzalez, D. Tetrahedron 1964, 20, 189-193.
Figure 2. (A) Depopulation of trans-azo-gA using UV irradiation. Absorption spectra illustrated the reduction in UV absorbance after illumination with 330-340 nm light from a Hg lamp for 90 min. (B) absorbance at 335 nm recorded at 5 min intervals during UV illumination. The decrease in UV absorbance corresponds to depopulation of the trans isomeric state. Single channel measurements were carried out using a commercial patch clamp amplifier (EPC7, List Medical, Darmstadt, F.R.G.) with a potential across the BLM of 100 mV. Data acquisition and preliminary analysis were carried out using an AMLAB data acquisition and processing module (Version 1.884, Associative Measurements, Chatswood, Australia). The data were filtered at 40 Hz. Exposure of the azo-gA to UV light was carried out in two ways: (i) The first were in situ, positioning a quartz optical fiber connected to a UV source, in front of the BLM and irradiating for between 30 and 60 min. Details of the BLM apparatus with its UV source are given in the Supporting Information (Figure 8). (ii) The second involves placing a quartz cuvette containing the gramicidin solution in front of the UV source for a period of 5-7 h. Following irradiation the gramicidin solution was injected into the BLM chamber. While recording measurements from dark-adapted samples, the laboratory was illuminated with a faint red light source. A 330-340 nm band-pass filter was used together with a 300 nm lower wavelength cut off to protect the tryptophans of the gA from the photolytic degradation that occurs below 290 nm.4,14
Results and Discussion Photoisomerization of the Azobenzene Moiety. UV-visible spectroscopy revealed that the azobenzene gA derivative in DMSO behaved much like other related azobenzenes, with an absorbance maximum at 338 nm and another weaker band at 434 nm. Figure 2 shows the UV spectra. The absorbance maximum at 338 nm (14) McKim, S.; Hinton, J. F. Biochim. Biophys. Acta 1993, 1153, 315-321.
4240 Langmuir, Vol. 14, No. 15, 1998
Osman et al.
Figure 4. Proton NMR spectra of the aromatic region of azobenzene (spectra on left) and azo-gA (spectra on right) in DMSO-d6. The top spectra show the dark-adapted or all-trans form of the molecules. The middle spectra are from the photostationary state after placing the samples in direct sunlight for 1 h. The bottom spectra were obtained after UV irradiation and are primarily the cis form of the molecules. Figure 3. (A) Repopulation of the trans isomeric state of azogA using elevated temperature. UV-visible spectra show the repopulation of the trans state resulting from elevating the sample temperature to 56 °C for 60 min. (B) repopulation of the trans state using blue irradiation, i.e., illumination at 430440 nm light from a Hg lamp for 90 min.
decreased and the band at 434 nm increased in intensity, as the trans state was converted to the cis isomer by irradiation at 330-340 nm. The photoisomerization could be reversed to give the trans isomer by illumination with blue light, or more effectively, by heating the samples in the dark at 333K for 1 h to give the dark-adapted trans state (Figure 3a,b). The photostationary state (trans:cis ratio of 3:1) could be achieved by exposure for several minutes to bright sunlight. The ratio of trans to cis isomers was confirmed by proton NMR (Figure 4). Initially, azobenzene derivatives including the carboxylic acid form were in the all-trans state when made up from solid powders and dissolved in DMSO. After exposure to sunlight, a 3:1 trans:cis photostationary state for the azobenzene was seen by NMR with the aromatic proton resonances at 7.5-8.0 ppm being due to the trans isomer and those at 6.7-7.4 ppm coming from the cis form. Selective irradiation at 330-340 nm increased the amount of cis isomer so that a ratio of
