Langmuir 2000, 16, 871-875
Interaction of Indolicidin with Model Lipid Bilayer: Quartz Crystal Microbalance and Atomic Force Microscopy Study
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aggregate that indolicidin molecules form upon incorporation into the lipid bilayer. 2. Experimental Section
Tai Hwan Ha, Chang Hwan Kim, Jong Sang Park, and Kwan Kim* Department of Chemistry and Center for Molecular Catalysis, Seoul National University, Seoul 151-742, Korea Received December 22, 1998. In Final Form: September 9, 1999
1. Introduction Ever since a tridecapeptide amide, indolicidin, was isolated by Selsted et al.1 from the cytoplasmic granules of bovine neutrophils, its biological activity has been studied extensively.2-4 The primary structure of indolicidin is H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-ArgArg-NH2. Indolicidin is known to possess antimicrobial activity,5,6 but its detailed mechanism is not yet understood clearly. The mode of action may be similar to that of other small peptides such as magainins, cecropins, and melittin. For the latter peptides, the antibiotic activity has been claimed to be due to the formation of ion channels or pores in the lipid membranes.7-9 On this ground, we have recently examined the interaction of indolicidin with model lipid bilayers using Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy.10 On the basis of the results of cyclic voltammetry (CV) measurements, indolicidin molecules were found to be incorporated inside the acyl chains of the lipid bilayers upon coming into contact with Langmuir-Blodgett (LB) lipid bilayers. In accordance with the circular dichroism (CD) measurements, it was also concluded that indolicidin, consisting of an ill-defined random coil structure in an aqueous medium, adopts a 310-helical conformation in LB lipid bilayers. It was very intriguing, however, that the perturbation of C-H stretching bands of model lipid molecules was not substantial even though indolicidin was incorporated clearly inside the lipid bilayers. We have performed in this work quartz crystal microbalance (QCM) and atomic force microscopy (AFM) studies, hoping to reveal how many indolicidin molecules are incorporated inside the lipid bilayer and to know the size of the * To whom all correspondence should be addressed. Telephone: 82-2-880-6651. Fax: 82-2-889-1568. E-mail: kwankim@ plaza.snu.ac.kr. (1) Selsted, M. E.; Novotny, M. J.; Morris, W. L.; Tang, Y. Q.; Smith, W.; Cullor, J. S. J. Biol. Chem. 1992, 267, 4292-4295. (2) Ahmad, I.; Perkins, W. R.; Lupan, D. M.; Selsted, M. E.; Janoff, A. S. Biochim. Biophys. Acta 1995, 1237, 109-114. (3) Aley, S. B.; Zimmerman, M.; Hetsko, M.; Selsted, M. E.; Gillin, F. D. Infect. Immun. 1994, 62, 5397-5403. (4) Abel, R. J. V.; Tang, Y.; Rao, V. S. V.; Dobbs, C. H.; Tran, D.; Barany, G.; Selsted, M. E. Int. J. Peptide Protein Res. 1995, 45, 401409. (5) Falla, T. J.; Karunaratnee, D. N.; Hancock, R. E. W. J. Biol. Chem. 1996, 271, 19298-19303. (6) Ladokhin, A. S.; Selsted, M. E.; White, S. H. Biophys. J. 1997, 72, 794-805. (7) Vaara, M.; Perro, M. Antimicrob. Agents Chemother. 1996, 40, 1801-1805. (8) Moore, A. J.; Beasley, W. D.; Bibby, M. C.; Devive, D. A. J. Antimicrob. Chemother. 1996, 37, 1077-1089. (9) Falla, T. J.; Hancock, R. E. W. Antimicrob. Agents Chemother. 1997, 41, 771-775. (10) Bahng, M. K.; Cho, N. J.; Park, J. S.; Kim, K. Langmuir 1998, 14, 463-470.
2.1. Materials. Indolicidin was prepared as reported previously.10 The synthetic phospholipid, that is, L-R-dipalmitoylphosphatidic acid (DPPA), was purchased from Sigma and stored below -5 °C before use. 1-Octadecanethiol (ODT) was purchased from Aldrich and used as received. All chemicals were reagent grade unless specified otherwise, and triply-distilled water (resistivity greater than 18.0 MΩ‚cm) was used throughout. 2.2. QCM Measurement. For the QCM measurement, titanium and gold (Aldrich, 99.99%) were evaporated consecutively onto an AT-cut quartz crystal (International Crystal Manufacturer, fundamental resonance frequency, f0 ) 10 MHz) at 1 × 10-6 Torr. The apparent area of the electrode was 0.2 cm2. The gold-coated quartz electrode thus prepared was immersed into a 5 mM ODT solution in ethanol for 12 h. After the electrode was removed, it was rinsed with excess ethanol and then dried with a N2 gas jet. Thereafter, a single DPPA layer was deposited onto the ODT layer by the LB method (via hydrophobic interaction, vide infra). The QCM apparatus consisted of the electrode thus obtained, a house-customized oscillator, and a frequency counter (Fluke PM6681) that was interfaced with a personal computer. The QCM cell, made of Teflon, was designed such that only the gold-coated side of the quartz was in contact with the solution. The other side was exposed to air to reduce the shunt effect, which might be significant when both sides were in contact with a polar medium.11,12 More details of the experimental setup are described elsewhere.13 We tested the QCM apparatus by monitoring the adsorption behavior of ODT on gold, and the data were in good agreement with the literature.11,14 For QCM measurement, 15 mL of 42 mM HEPES buffer at pH 7 was added to the cell, and then the system was allowed to stabilize under stirring with a small bar magnet. In a few minutes, the measured frequency was stabilized within a range of 1-2 Hz. Thereafter, 10 µL of indolicidin stock solution was added to the cell with a microsyringe to the final concentration 30 µg/mL, and frequency changes of the crystal were monitored as a function of time. 2.3. LB Films. For the LB deposition of DPPA either on an ODT layer or on freshly cleaved mica (Asheville-Shoonmaker), DPPA (0.5 mg/mL in chloroform) was spread on the air-water interface of the Langmuir trough (KSV Model 3000) thermostated at 18 °C, and CHCl3 was allowed to evaporate for 1 h. The mica substrate had previously been immersed into the subphase of water. The DPPA layer was compressed at a rate of 2 mm/min to a surface pressure of 40 mN/m. A Y-type deposition15 was employed to anchor lipid single bilayers onto the hydrophilic mica (for AFM measurement, vide infra) by raising and lowering the mica vertically at the rates 2 and 4 mm/min, respectively. The intermediate drying time was at least 70 min at 50-60% humidity. With a short drying time, the second DPPA layer was easily separated from the first layer when taking out the LB film from the water subphase. To deposit a single DPPA layer onto the ODT/Au-covered quartz electrode16 (for QCM measurement, vide supra), the substrate positioned vertically outside the water was lowered into the water subphase at 40 mN/m at the rate 4 mm/min. The DPPA-covered mica and the quartz substrates were finally removed from the water subphase after the DPPA molecules remaining at the air-water interface had been completely sucked out by an aspirator. (11) Karpovich, D. S.; Blanchard, G. J. Langmuir 1994, 10, 33153322. (12) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1356-1379. (13) Ha, T. H. M.S. Dissertation, Seoul National University, Seoul, Korea, 1998. (14) Okahata, Y.; Matsunobu, Y.; Ijiro, K.; Mukae, M.; Murakami, A.; Makino, K. J. Am. Chem. Soc. 1992, 114, 8299-8300. (15) Bassereau, P.; Pincet, F. Langmuir 1997, 13, 7003-7007. (16) Meuse, C. W.; Niaura, G.; Lewis, M. L.; Plant, A. L. Langmuir 1998, 14, 1604-1611.
10.1021/la981752y CCC: $19.00 © 2000 American Chemical Society Published on Web 10/28/1999
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revealed that the final frequency change is also barely dependent on the temperature of the indolicidin solution in the range 25-37 °C. The degree of incorporation of indolicidin into DPPA may be estimated from the frequency change. For this purpose, we assumed the Sauerbrey equation17 to hold in our system:
∆f ) -
Figure 1. Frequency change of QCM in a 30 µg/mL indolicidin solution. The arrow indicates the time at which an indolicidin stock solution was injected into the cell. The QCM electrode was previously coated consecutively with ODT and DPPA layers. The time interval to reach the plateau and the total frequency change were nearly invariant in the concentration range 3050 µg/mL. 2.4. AFM Measurement. AFM images of the DPPA-covered mica substrates were obtained with a Digital Instruments Nanoscope IIIa scanning probe microscope. After the DPPAcovered mica substrate was positioned inside a liquid cell (Digital Instruments), the cell was filled with an aqueous 42 mM HEPES buffer at pH 7. Using a V-shaped 200 µm long Si3N4 cantilever with a nominal spring constant of 0.12 N/m (Nanoprobe, Digital Instruments), topographic images were recorded in the tapping mode with a drive frequency of ∼30 kHz at certain time intervals after the injection of indolicidin into the cell to a final concentration of 30 µg/mL. Images were obtained at a scan rate of 2-3 Hz. The AFM and QCM measurements were performed at room temperature.
3. Results and Discussion Our earlier CV measurements and the present QCM and AFM measurements were all performed in aqueous media with a DPPA layer assembled previously on gold or mica substrates. In contrast, the FTIR-ATR spectra were taken earlier under ex-situ conditions after soaking the DPPA-coated substrate in an aqueous solution of indolicidin. However, considering that some water molecules inevitably exist on the DPPA layer in a laboratory environment, the FTIR-ATR spectroscopic data may also be related to the QCM and AFM data presented below. 3.1. QCM Observation. Figure 1 shows the QCM data observed after the DPPA/ODT/Au-coated quartz was placed in contact with a 30 µg/mL indolicidin solution. Upon injection of the indolicidin solution, the resonance frequency of quartz abruptly decreased and was stabilized after ∼15 min. This implies that the interaction of indolicidin with DPPA occurs very rapidly, in agreement with the previous FTIR-ATR observation.10 The total frequency change was nearly invariant in the concentration range 30-50 µg/mL (data not shown), and the time interval to reach the plateau also did not exceed 15 min. It has to be mentioned that, in the previous work, the DPPA layer was maintained in contact with the indolicidin solution at 37 °C, and then the FTIR-ATR and CV measurements were performed under ex-situ conditions at room temperature. The present in-situ QCM measurement is made, however, at room temperature (ca. 25 °C), so the rate of the incorporation of indolicidin into the DPPA layer in this work may not be the same as that in the previous work. Nonetheless, separate ex-situ QCM data
2f02∆m µq1/2Fq1/2A
(1)
in which ∆f is the amount of frequency change resulting from a change in mass (∆m) by incorporation of indolicidin into DPPA, f0 is the fundamental resonance frequency of the quartz crystal employed (10 MHz), µq is the shear modulus of quartz (2.947 × 1011 dyn/cm2 for an AT-cut quartz), Fq is the density of quartz (2.648 g/cm3), and A is the geometric area of the metal-coated quartz. In the liquid-phase experiment, the QCM data should be interpreted carefully to include the fluid effect on the piezoelectric movement of quartz.12,14 When a metal part is in contact with a solution, the piezoelectric movement is usually damped, resulting in a decreased frequency. Such a frequency change can be ignored, however, if both the initial and final states are under nearly the same environment with respect to the density, viscosity, and temperature of the solution phase. In fact, several examples are available in the literature showing that the Sauerbrey equation is obeyed in a solution phase, particularly when rigid films such as lipid bilayers are deposited on metal-coated quartz electrodes.12,18 When the frequency change measured at the plateau region, ∆fmax (-30 ( 7 Hz), was substituted into eq 1, the maximum mass increase of 133 ng/cm2 was obtained. Recalling the molecular mass of indolicidin (MW 1903.6 g/mol) and the apparent surface area of the gold-coated quartz (0.20 cm2), the latter value should correspond to a surface coverage of 7.0 × 10-11 mol/cm2. Considering that, in the surface pressure-area isotherm of DPPA at the air-water interface at 18 °C, the limiting surface area of DPPA is determined to be 42 ((1) Å2, the above surface coverage corresponds to the incorporation of one indolicidin molecule per 5.7 DPPA molecules on the average. (Referring to the report of Besenhard et al.,19 we determined the roughness factor of the gold-coated quartz by an electrochemical method; the ratio of the actual to geometric area was 1.2 ( 0.2. Assuming a roughness factor of 1.2, one indolicidin molecule might have been incorporated into the lipid layer per 6.8 DPPA molecules.) 3.2. AFM Observation. Having confirmed the interaction of indolicidin molecules with the DPPA layer by QCM, the morphological changes of the DPPA layer were examined by an in-situ AFM measurement. Figure 2a represents a typical AFM image of a DPPA single bilayer deposited on mica by the LB method. The image corresponds to the morphology of the QCM sample marked as “image 1” in Figure 1. The DPPA layer is in fact quite homogeneous, taking into account that some defect sites, that is, pits, inevitably exist when the LB method is applied to the assembly of the lipid molecules.20-22 Such a pit could be used as a marking point for subsequent AFM mea(17) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (18) Ebara, Y.; Itakura, K.; Okahata, Y. Langmuir 1996, 12, 51655170. (19) Besenhard, J. O.; Parsons, R.; Reeves, R. M. J. Electroanal. Chem. 1979, 57-72. (20) Egger, M.; Ohnesorge, F.; Weisenhorn, A. L.; Heyn, S. P.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Gaub, H. E. J. Struct. Biol. 1990, 103, 89-94.
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Figure 2. (a) AFM image (3.0 µm × 3.0 µm) of a single DPPA LB bilayer on mica recorded in the tapping mode in aqueous HEPES solution at pH 7. Several pits are formed when the LB deposition is applied. (b) AFM image of the DPPA bilayer taken 3 min after injection of indolicidin. The area scanned in part b is different from that in part a due to the turbulence in the sample solution induced by injection of indolicidin. (c) AFM image of the DPPA bilayer taken 7 min after injection of indolicidin. The pits seen in part b are used as the marker to image the same location.
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Figure 3. Structures of (a) 310- and (b) R-helical conformations of peptides.
surements. The AFM image became noisier upon injection of indolicidin. Probably due to the induced turbulence in the sample solution, the AFM tip could no longer be located at the same area as that in Figure 2a. Nonetheless, about 2 min after injection of indolicidin, the solution phase appeared to be fairly stabilized. Figure 2b shows the AFM image of a DPPA bilayer taken 3 min after injection of the indolicidin molecules. The image corresponds to the morphology of the QCM sample marked as “image 2” in Figure 1. Many dark areas are clearly seen in Figure 2b, illustrating formation of deep holes by the action of indolicidin with DPPA. As time goes on, the morphology gets rougher and then suddenly becomes smooth and homogeneous. This is evident from the AFM image in Figure 2c taken 7 min after injection of the indolicidin molecules. The image in Figure 2c was taken from the same location as that in Figure 2b but different from that in Figure 2a. The image in Figure 2c could be regarded as reflecting the morphology of the QCM sample marked as “image 3” in Figure 1. In fact, the present AFM observation seems to be consistent with our earlier CV measurement.10 When a DPPA/ODT-coated Au electrode which had been soaked previously in indolicidin solution for 15 min was put in an electrolyte containing Fe(CN)63-, cathodic and anodic peaks of Fe(CN)63- were clearly identified. However, using a DPPA/ ODT-coated Au electrode soaked previously in indolicidin solution for >30 min, CV peaks were not identified at all. It was thus supposed that the indolicidin molecules that penetrated the lipid bilayer eventually assembled along with the lipid molecules nearby, forming a rather closepacked structure that does not allow passage of electroactive species through the film. 3.3. Model Scheme of Interaction between DPPA and Indolicidin. In planning the AFM measurement, we hoped to reveal the size of aggregates of indolicidin molecules formed upon incorporation into the lipid bilayers. To prevent any tip-induced morphological change, we employed a tapping mode operation in the AFM measurement. Using this method, we could not obtain AFM images on a nanometer scale, but the dark areas in Figure 2b as well as their subsequent disappearance in Figure 2c were reproducibly observed irrespective of the scan direction, at least on a micrometer scale. The holes (dark areas) identified in Figure 2b are distributed with sizes ranging from 30 to 100 nm. Unfortunately, the depth of the holes could not be measured in tapping mode, probably due to the finite size of the AFM tip used. Although the identity of the holes is a matter of conjecture at the moment, they may represent canals created by aggregation of indolicidin (21) Weisenhorn, A. L.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Ohnesorge, F.; Egger, M.; Heyn, S. P.; Gaub, H. E. Biophys. J. 1990, 58, 1251-1258. (22) Zasadzinski, J. A. N.; Helm, C. A.; Longo, M. L.; Weisenhorn, A. L.; Gould, S. A. C.; Hansma, P. K. Biophys. J. 1991, 59, 755-760.
Figure 4. Plausible model for explaining the AFM images in Figure 2b and c: (a) 3 min and (b) 7 min after injection of indolicidin.
molecules, as in the case of other small peptides such as magainins, cecropins, melittin,9 and gramicidins.23,24 (In our previous IR spectroscopy and CD studies,10 indolicidin was concluded to adopt a 310-helical conformation in LB lipid bilayers; see Figure 3 for the 310-helical conformation. The size of the holes in Figure 2b is obviously >20 times larger than the cross-section of a single 310-helical strand.) On the other hand, the holes may reflect the vacancies created by DPPA molecules desorbed from the lipid bilayer by the action of indolicidin. To see the feasibility of desorbing DPPA molecules, we performed a separate QCM experiment using a Au-coated quartz onto which only a single ODT layer was deposited beforehand. Upon injection of indolicidin solution, the resonance frequency of quartz abruptly decreased and was stabilized after ∼15 min, as was the case in Figure 1. Moreover, the amount of change in frequency after 15 min was about -30 Hz. The change in frequency is almost the same as that observed with the DPPA/ODT/Au-coated quartz. Considering that the Au-S bond is rather strong, ODT molecules will hardly be desorbed from the Au surface even by the action of indolicidin. This implies that the substantial frequency change must be associated with the hydrophobic interaction between ODT and indolicidin molecules. On the other hand, since indolicidin is a cationic peptide,6 its interaction with DPPA is presumably stronger than that with ODT because the former interaction is both ionic and hydrophobic. The comparable frequency change in two different QCM experiments seems to imply that some DPPA molecules can be desorbed from DPPA layers on quartz by the action of indolicidin molecules. On the basis of the above arguments, we speculate that various holes in Figure 2b are associated with desorption of some DPPA molecules rather than formation of canals by indolicidin molecules. This scheme of interaction between DPPA and indolicidin is in fact quite similar to (23) Ulrich, W.-P.; Vogel, H. Biophys. J. 1999, 76, 1639-1647. (24) Nelson, A. Langmuir 1996, 12, 2058-2067.
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the action mechanism of gramicidin S and melittin on human erythrocytes reported by Katsu et al.25 In this light, the plausible explanation of the situation in Figure 2b is illustrated in Figure 4a. We speculate further that, due to a rather strong interaction between DPPA and indolicidin, indolicidin molecules remaining in the aqueous medium are readily incorporated inside the holes of DPPA layers on the mica substrate, resulting in the disappearance of holes in Figure 2c. In fact, the height of the DPPA layer in the AFM image in Figure 2c was determined to be ∼5 nm with a contact mode measurement. This imples that a bilayer structure is maintained for DPPA on mica. On these grounds, the plausible illustration of the situation in Figure 2c is shown in Figure 4b. 4. Summary and Conclusion Through in-situ QCM and AFM experiments we obtained detailed information about the interaction of indolicidin molecules with lipid membranes. We confirmed from QCM experiment that the interaction of indolicidin with DPPA occurs very rapidly. From the AFM experiment, numerous holes ranging from 30 to 100 nm were observed to form initially on the lipid layer by the action of indolicidin. As time went on, the morphology of the DPPA layer became, however, smooth and homogeneous. Although the detailed nature of the interaction of indolicidin with DPPA molecules is still uncertain, the initial (25) Katsu, T.; Kuroko, M.; Morikawa, T.; Sanchika, K.; Fujita, Y.; Yamamura, H.; Uda, M. Biochim. Biophys. Acta 1989, 983, 135-141.
formation of holes is supposed to arise from desorption of DPPA molecules from the underlying mica substrate by the action of indolicidin. With the help of stronger hydrophobic interaction between the DPPA and indolicidin molecules, the damaged DPPA layer might be remedied by the incorporation of indolicidin molecules in the aqueous medium into the holes. The present interpretation seems to support the earlier view that the aggregated indolicidin should eventually assemble along with the nearby lipid molecules into a close-packed structure. Nonetheless, further studies are needed to elucidate the mechanism of hole formation at the model lipid membrane. We plan to investigate the effect of temperature, pH, and indolicidin concentration on the size of holes to obtain insights into the aggregate structure and the nature of association. In addition, we hope to see whether the hole sizes will change for other model lipid bilayers such as DPPC. It is desirable to perform an isotope-labeling experiment to confirm the feasibility of desorption of lipid molecules by indolicidin molecules. Acknowledgment. This work was supported by the Korea Science and Engineering Foundation through the Center for Molecular Catalysis at Seoul National University (SNU) and by the Korea Research Foundation through the Research Institute for Basic Sciences at SNU. K.K. appreciates several helpful comments from Professor Hie Joon Kim in the Chemistry Department of SNU. LA981752Y
