Peripheral and Integral Membrane Binding of Peptides Characterized

(22) The pH-dependent antimicrobial and nucleic acid transfection activities are of .... at pH 8, 4.5, or 7.4 prepared with filtered water (Milli-Q3 s...
0 downloads 0 Views 514KB Size
Download PDF
Article pubs.acs.org/Langmuir

Peripheral and Integral Membrane Binding of Peptides Characterized by Time-Dependent Fluorescence Shifts: Focus on Antimicrobial Peptide LAH4 Radek Macháň,† Piotr Jurkiewicz,† Agnieszka Olzẏ ńska,† Marie Olšinová,† Marek Cebecauer,† Arnaud Marquette,‡ Burkhard Bechinger,‡ and Martin Hof*,† †

J. Heyrovský Institute of Physical Chemistry of ASCR, v.v.i., Dolejškova 3, Prague 8, CZ-18223, Czech Repulic Université de Strasbourg/CNRS, UMR7177, Institut de Chimie, 1 rue Blaise Pascal, 67000 Strasbourg, France

‡

ABSTRACT: Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.

■

INTRODUCTION Antimicrobial peptides have been attracting attention for years as prospective antibiotics. A variety of peptides have been isolated from natural sources, and many more have been designed.1,2 Linear cationic amphipathic peptides are especially interesting since they can be synthesized in sufficient quantities for biological and biophysical investigations,3,4 and the frequently studied members of this peptide class, magainins and cecropins, display high antimicrobial efficiency and selectivity.5,6 The most significant common feature shared by peptides belonging to this class is the amphipathic distribution of polar and hydrophobic residues determining their capacity to interact with phospholipid membranes. They typically disturb the bilayer integrity by the formation of defects and pores or by detergent-like effects resulting in the loss of transmembrane electrochemical gradients of the target cell.7−9 Such effects may explain the antibiotic activities of the peptides, although it is possible that the action of some peptides involves intracellular targets as well.10 It has been shown that neither the details of the amino acid sequence nor the chirality significantly influence the antimicrobial activity of the peptides as long as a net © 2014 American Chemical Society

positive charge and the ability to form amphipathic structures at membrane interfaces are maintained.11−13 It is, therefore, obvious that the antimicrobial activity is not mediated via protein receptors and that the direct interaction of the peptides with membranes is essential for their activity. To understand how the physical characteristics of peptide molecules such as the net positive charge, hydrophobicity, and hydrophobic moment determine the antibiotic activity as well as selectivity of the peptides, model molecules with well-defined properties have been designed.14−17 LAH4 is an example of such a model peptide. Its name is derived from its amino acid sequence composed of leucines, alanines, and four histidines; there are also two pairs of lysines located at both ends. The sequence has a high propensity to form α-helical structures in membrane environments, and the histidines allow the manipulation of the net charge and hydrophobic moment of the helix by merely changing the pH.18 It has been shown on Received: February 17, 2014 Revised: April 11, 2014 Published: May 7, 2014 6171

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179

Langmuir

Article

fluorenylmethyloxycarbonyl) chemistry. A 4-fold excess of Fmocprotected amino acids (Bachem, Heidelberg, Germany and Applied Biosystems, Weiterstadt, Germany) was used during chain elongation. After TFA (trifluoroacetic acid) cleavage, the peptide was purified by preparative HPLC using an acetonitrile/water gradient in the presence of 0.1% TFA and a 214 nm detection wavelength. The identity and high purity of the product (>90%) were confirmed by MALDI mass spectrometry and analytical HPLC. Peptides LW21 ((MW 4120, Gly-Leu-Leu-Asp-Ser-(Lys)2-(Trp)2(Leu)8-Ala-(Leu)8-(Trp)2-(Lys)2-Phe-Ser-Arg-Ser-NH2) and LAT (MW 3236, Glu-Glu-Ala-Ile-Leu-Val-Pro-Ser-Val-Leu-Gly-(Leu)4Pro-Ile-Leu-Ala-Met-Leu-Met-Ala-Leu-Ser-Val-His-Ser-His-Arg-NH2) were purchased from VIDIA (Prague, Czech Republic). The identity and purity of the products (∼80%) were confirmed by MALDI mass spectrometry and analytical HPLC. The sequence of LW21 peptide contains 21 hydrophobic residues of the original LW peptide flanked by two lysine residues and a native sequence of the membrane proximal motif from human TCRζ (five N- and four C-terminal residues). Similarly, the LAT peptide contains all residues of the putative transmembrane domain (TMD) of human LAT protein flanked by the membrane proximal motif (three N- and four Cterminal residues). Solutions of 100 μM LW21 and 86 μM LAT peptides were freshly prepared in TFE for each experiment. All experiments were done in 100 mM phosphate buffer at pH 8, 4.5, or 7.4 prepared with filtered water (Milli-Q3 system, Millipore, Etten-Leur, The Netherlands). The buffer for fluorescence correlation spectroscopy (FCS) contained 1 mM ethylenediaminetetraacetic acid (EDTA) to prevent the aggregation of vesicles caused by trace amounts of divalent cations or their interactions with peptides. Large Unilamellar Vesicles (LUVs). Liposomes of 100 nm diameter were prepared as described below. Lipids dissolved in chloroform and optional fluorescent probes dissolved in methanol were mixed in the desired ratio, and the solvent was removed by a stream of nitrogen; dry lipid films were hydrated in buffer for more than 30 min (to 2 mM total lipid concentration), and the suspension was extruded (at least 30 times) through a polycarbonate filter (Avestin, Ottawa, Canada) of 100 nm pore size. LUVs for the solvent relaxation study contained Laurdan (1:100 mol/mol probe/lipid), and LUVs for the FCS study of LUV diffusion contained BODIPY FL DHPE (1:500 mol/mol probe/lipid). LUVs for the dye leakage essay were prepared in a buffer containing 4 μM Alexa 488 and after extrusion were dialyzed through a cellulose membrane with a 14 kDa cutoff (Visking, London, U.K.) against 500 mL of dye-free buffer; the buffer was exchanged three times. Generalized Polarization (GP). The excitational generalized polarization as defined in Parasassi et al.29 was calculated

supported bilayers that LAH4 helices are oriented parallel to the membrane surface at pH lower than approximately 6, when the histidines carry net positive charges and adopt transmembrane orientation at neutral and basic pH.15,19 Apart from being an interesting model peptide whose charge and hydrophobic moment can be changed without altering its amino acid sequence, LAH4 and derivatives thereof are also efficient antimicrobial peptides with a stronger effect against some strains compared to magainin 2.20 Interestingly, an acidic pH enhances its antimicrobial activity.20,21 Additionally, the other known biological activities of LAH4, namely the DNA and RNA transfection of eukaryotic cells, are also modulated by changes in pH.22 The pH-dependent antimicrobial and nucleic acid transfection activities are of practical importance, for example, in development of the treatment of cystic fibrosis.21 LAH4 has also been shown to facilitate the intracellular delivery of protein-based vaccines adjuvanted with CpG oligonucleotides23 and an LAH4 derivative to increase the lentiviral transduction of human hematopoietic stem cells.24 In the present study, we combined two fluorescence techniques, namely, the time-dependent fluorescence shift method (TDFS) and fluorescence correlation spectroscopy (FCS), to investigate the interactions of model amphiphilic peptide LAH4 with large unilamellar vesicles. TDFS using Laurdan characterizes the polarity and mobility on the glycerol level.25,26 Alongside the experiments for LAH4 at acidic and basic pH, analogous experiments were performed for antibacterial peptides alamethicin and magainin 2 (known to bind to membranes in a transmembrane and peripheral manner, respectively)7 as well as for synthetic TMPs LW21 and LAT.27,28 The results obtained for these four peptides provide support for our interpretation of the results achieved using LAH4 in terms of the orientation of peptide molecules. We are showing here, according to our knowledge for the first time, how time-dependent fluorescence shifts can be employed to estimate the orientation of a peptide with respect to the membrane. Furthermore, FCS provides information on the peptide-induced membrane permeation and stability of phospholipid vesicles under conditions in which permeation occurs. In contrast to previous solid-state NMR and ATR-FTIR investigations, which have been performed on supported lipid bilayers and in the absence of bulk water, the fluorescence methods presented here allow the characterization of the freestanding fully hydrated lipid bilayers that are closer to physiological conditions.

■

GPEX =

I440 − I490 I440 + I490

(1)

where I440 and I490 represent the fluorescence intensities emitted at 440 and 490 nm, respectively (excited at 373 nm). Steady-state emission spectra for GP and also for the TDFS method were collected using a Fluorolog-3 spectrofluorimeter (model FL3-11, Jobin Yvon Inc., Edison, NJ, USA) equipped with a xenon arc lamp. Time-Dependent Fluorescence Shift (TDFS). A detailed description of the method can be found elsewhere.26 Briefly, timeresolved emission spectra (TRES) obtained by the spectral reconstruction method were fitted to log-normal functions in order to determine the positions of their maxima ν(t) and their width (full width at half-maximum). The correlation function defined by eq 2 was calculated by taking the estimated time-zero spectrum as ν(0).26

EXPERIMENTAL SECTION

Chemicals. The lipids, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The headgroup-labeled phospholipid N-(4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (BODIPY FL DHPE), Alexa Fluor 488 C5 maleimide (Alexa 488), and 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) were ordered from Invitrogen (Carlsbad, CA). All other chemicals, including peptides alamethicin (Ac-Aib-Pro-Aib-AlaAib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phl from Trichoderma viride) and magainin 2 (MW 2467, Gly-Ile-Gly-LysPhe-Leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-Glu-IleMet-Asn-Ser), were purchased from Sigma-Aldrich. The peptide LAH4 (MW 2777, Lys-Lys-Ala-Leu-Leu-Ala-Leu-AlaLeu-His-His-Leu-Ala-His-Leu-Ala-Leu-His-Leu-Ala-Leu-Ala-Leu-LysLys-Ala-NH2) was prepared by solid-phase peptide synthesis on Millipore 9050 automatic peptide synthesizer and Fmoc (9-

C(t ) =

v(t ) − v(∞) v(t ) − v(∞) = v(0) − v(∞) Δv

(2)

To quantify the relaxation process, two parameters are determined. The first one, Δν, represents the overall emission shift, which is directly proportional to the polarity of the dye environment. (For illustration, see Figure 9 in the seminal work of the Maroncelli group.30) Since in a pure phospholipid bilayer the polarity is mainly 6172

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179

Langmuir

Article

determined by water molecules, Δν reflects the extent of membrane hydration.26 In the case investigated here, in which polar residues of the peptides might influence the polarity at the glycerol level of the bilayer, we prefer to use the more general term polarity. The second parameter, relaxation time τr, describes the mobility of the dye environment. In the phospholipid bilayer at the level of glycerol, where Laurdan is located, water hydrating the membrane is fully bound to the phospholipid molecules as their solvation envelopes.26 Therefore, the relaxation kinetics observed in membranes is attributed to the collective relaxation of the dye environment and reflects local membrane dynamics rather than the motions of water molecules alone. The so-called integrated solvent relaxation time is defined by eq 3. The intrinsic uncertainty of this parameter was assumed to be ∼20 ps, based on the time resolution of the experimental setup.

τr =

∫0

⎡ ⎛ 1 1 τ ⎞⎤ G(τ ) = 1 + ⎢1 − T + T exp⎜ − ⎟⎥ ⎢⎣ ⎝ τT ⎠⎥⎦ N (1 − T ) 1 +

( )

⎛ ⎜ 1 ⎜ ⎜ 1 + τ S2 τD ⎝

( )

⎞ ⎟ ⎟ ⎟ ⎠

(4)

If the model (eq 4) was not sufficient to fit the autocorrelation function, then the model (eq 5) with two diffusion times was applied.36,37 ⎡ ⎛ 1 τ ⎞⎤ G(τ ) = 1 + ⎢1 − T + T exp⎜ − ⎟⎥ ⎢⎣ τ ⎝ ⎦ (1 − T ) T ⎠⎥ ⎡ ⎢ A ⎢ ⎢1 + τ τDA ⎢⎣

∞

C(t ) dt

τ τD

( )

(3)

1 1+

( S) τ 2 τDA

B

+ 1+

( ) τ τDB

⎛ ⎜ 1 ⎜ ⎜ 1 + τ S2 τDB ⎝

( )

⎞⎤ ⎟⎥ ⎟⎥ ⎟⎥ ⎠ ⎥⎦

(5) Amplitudes A and B are related to N, the fraction FA of particles moving with diffusion time τDA and the ratio of molecular brightness QA and QB by (eqs 6)

Fluorescence decays were recorded on a 5000U single photon counting setup (IBH, Glasgow, U.K.) using an IBH laser diode NanoLED 11 (370 nm peak wavelength, 80 ps pulse width, 1 MHz maximum repetition rate) and a cooled Hamamatsu R3809U-50 microchannel plate photomultiplier. Emission decays were recorded at a series of wavelengths spanning the steady-state emission spectrum (400−540 nm) in 10 nm steps (emission slits: 8 nm bandwidths) at magic angle polarization. Additionally, a 399 nm cutoff filter was used to eliminate scattered light. The signal was kept below 2% of the light source repetition rate (1 MHz). Data were collected in 8192 channels (0.014 ns per channel) until the peak value reached 5000 counts. Fluorescence decays were fitted to multiexponential functions using the iterative reconvolution procedure using IBH DAS6 software. The fitted decays together with the steady-state emission spectrum were used for the reconstruction of TRES.30 TDFS experiments were performed at 20 °C for POPC LUVs suspended in a buffer. LAH4, alamethicin, and magainin 2 were added to the LUV suspension to reach the desired peptide to lipid ratio (in the range from 0 to 1:10). LW21 and LAT, due to their low solubility in water, were added to the dye/lipid solution in the organic solvents during LUV preparation. The measurements were performed at pH 4.5 or 8.0 for LAH4 and at pH 7.4 for magainin 2, alamethicin, LW21, and LAT. Please note that the peptide to lipid ratios given in this work are the ratios of their total concentrations in the samples and that depending on the binding affinity of the particular peptide the bound peptide to lipid ratio may differ. Fluorescence Correlation Spectroscopy (FCS). Measurements were performed using a Confocor 1 (Carl Zeiss, Jena, Germany; Evotec Biosystems, Hamburg, Germany) confocal microscope with a pulsed 470 nm diode laser (PicoQuant, Berlin, Germany) as an excitation source. The laser power was kept below 10 μW to minimize photobleaching and saturation.31 The time trace of fluorescence intensity was recorded for 300 s by a TimeHarp 200 data acquisition card and processed in Origin (OriginLab, Northhampton, MA) as described elsewhere.32 All measurements were performed in 8-well Lab-Tek chambers (Nalge Nunc, Rochester, NY). The resulting autocorrelation functions of fluorescence intensity fluctuations were fitted with a model for 3D free diffusion (eq 4), where N is the number of independently diffusing particles within the effective detection volume, diffusion time τD is the characteristic time spent by a diffusing particle within the detection volume, τT is a characteristic time scale of fluorophore transition to the triplet state, T is the average fraction of fluorophores in the triplet state, and S is a parameter describing the shape of the detection volume.33−35 On the basis of calibration measurements with an aqueous solution of Alexa 488, the value of S was set to 7 and τT to 50 μs; τT of BODIPY FL DHPE was set to 1 μs.

A=

Q A 2FA N (Q AFA + Q B(1 − FA))2

B=

Q B2(1 − FA) N (Q AFA + Q B(1 − FA))2

(6)

Model (eq 5) is essential for the dye leakage assay characterized by FCS.36−38 The sample in that case contains a mixture of free dye molecules moving with diffusion time τDB of a free Alexa 488 molecule and dye molecules entrapped within vesicles moving with diffusion time τDA of a 100 nm vesicle. The sample is titrated with a peptide solution, and if the peptide causes dye leakage, the fraction Fent of dye molecules entrapped in vesicles is decreasing with an increasing peptide to lipid ratio. Fent can be expressed using eqs 6 and the average number of dye molecules within a vesicle m = QA/QB. m Fent = mFA = B 1 + m2 A (7) Since the average total number M of dye molecules within the detection volume (M = N[FA(m − 1) + 1]) remains constant throughout the whole titration experiment, eqs 6 and 7 give a linear relationship (eq 8) between amplitudes A and B. The value of m can be thus determined for each vesicle preparation from the relation between experimentally obtained amplitudes A and B, and using eq 7, we arrive at the value of Fent for each peptide to lipid ratio.

■

⎛ 1 ⎞ ⎟ A = m⎜ ⎝ M − B⎠

(8)

RESULTS AND DISCUSSION Time-Dependent Fluorescence Shift: Does LAH4 Bind to Neutral LUVs in a Peripheral or Transmembrane Manner? Measurements using Laurdan provide information on polarity and mobility at the glycerol level of the bilayer.26 The location of Laurdan within a lipid bilayer is shown in Figure 1. The influence of LAH4 binding on the probed mobility (τr) and polarity (Δν) in zwitterionic POPC LUVs was investigated using the time-dependent fluorescence shift (TDFS) method. To support our interpretation of the results in terms of the orientation of LAH4 molecules, we performed analogous experiments for magainin 2 (peripheral binding) and alamethicin, LW21, and LAT (transmembrane insertion). All 6173

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179

Langmuir

Article

Figure 1. Chemical structure and location26 of Laurdan in a POPC bilayer.

Figure 2. Relative relaxation time measured for Laurdan in POPC LUV suspension using the TDFS method. τr and τr0 are integrated relaxation times measured in the presence and absence of peptide, respectively. The absolute values can be found in Table 1. Samples with LAH4 were measured at pH 4.5 and 8.0; samples with magainin 2, alamethicin, LW21, and LAT, at pH 7.4.

of the results are summarized in Table 1. Considering the widespread use of Laurdan generalized polarization (GP),39 we add the corresponding values. The most significant and meaningful changes (τr measured for Laurdan) are depicted in Figure 2 in a relative fashion, i.e., as a ratio between τr values measured in the presence and in the absence of the peptide. LAH4 has been previously shown by NMR experiments to bind to supported lipid bilayers at pH 4.5 in a peripheral manner.15,19 The solvent relaxation time, τr, and the Δν values (providing information on mobility and polarity at the glycerol level) reported by Laurdan are very little influenced by LAH4 binding at this pH (Table 1), indicating that the acyl groups about 1 nm below the external interface of the bilayer26 are not really “feeling” the peripherally bound peptide. Analogous experiments with magainin 2 show changes in τr and Δν which are also close to or within the experimental error (the same is true for the GP values). We conclude that the peripheral

binding of these α-helical peptides does not influence the polarity and mobility on the glycerol level of the bilayer. When experiments were performed at pH 8, the influence of peptide on membrane properties, as sensed by Laurdan, was very different. A 40% increase in τr due to the presence of LAH4 at a 10:1 lipid to protein ratio gives strong evidence for the reduced mobility of lipid acyl groups. On the other hand, the probed polarity (Δν) appears to be constant within the experimental error. It should be noted that in a series of experiments performed on lipid bilayers (in the absence of peptides) such a substantial increase in τr is, as a rule, connected to a significant decrease in Δν, for example, decreases in hydration and mobility due to a decrease in the temperature above the main lipid phase transition (e.g., see Table 1 in Beranova et al.40) or due to the addition of alkali cations to phospatidylserine-containing bilayers (see Table 1 in

Table 1. Polarity (Δν), Mobility (τr), and Generalized Polarization (GPEX) Detected by Laurdan Located at the Glycerol Level in Zwitterionic POPC LUVs (1:100 Dye/Lipid Mol/Mol Ratio)a Laurdan peptide

pH

peptide/lipid [mol/mol]

− LAH4 LAH4 − LAH4 LAH4 − magainin 2 magainin 2 alamethicin alamethicin LAT LAT LAT LW21 LW21 LW21

4.5 4.5 4.5 8.0 8.0 8.0 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4

0 1:100 1:10 0 1:100 1:10 0 1:100 1:10 1:100 1:10 1:100 1:50 1:10 1:100 1:50 1:10

Δν [cm−1] 3980 3980 3960 4040 4010 4060 4067 4060 4020 4040 4020 4090 4120 4110 4190 4150 4180

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

60 60 50 60 50 50 50 50 50 50 50 50 50 50 50 50 50

τr [ns] 1.86 1.83 1.86 1.62 1.70 2.35 1.59 1.57 1.66 1.81 2.82 1.72 1.78 1.88 1.58 1.75 2.77

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.04 0.02 0.07 0.02 0.03 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

GPEX −0.127 −0.138 −0.126 −0.161 −0.152 −0.073 −0.159 −0.188 −0.154 −0.142 −0.018 −0.126 −0.125 −0.096 −0.158 −0.119 −0.020

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.017 0.018 0.017 0.021 0.020 0.021 0.021 0.024 0.020 0.019 0.020 0.017 0.017 0.013 0.021 0.016 0.020

Measurements were performed at 20 °C and pH 4.5 and 8.0 for LAH4 and pH 7.4 for magainin 2, alamethicin, LW21, and LAT. Δν, τr, and GPEX parameters are defined in the Experimental Section. a

6174

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179

Langmuir

Article

Jurkiewicz et al.41). Naturally the GP values increase in such cases. For the experiment on peptides presented here, the substantial increase in τr together with unchanged Δν indicate that the hindered rotational and translational freedom of the acyl groups cannot be simply connected to a less hydrated (and thus less mobile) glycerol level. In order to explain the observed changes, it should be considered that in case of peptide insertion Δν does not necessarily describe only the hydration but can also be affected by the presence of the peptide in the vicinity of the probe. Similar trends in membrane properties were sensed by Laurdan when alamethicin was added to the LUVs (Figure 2), with the increase in τr being even larger in this case. In both cases, the TDFS experiments show that LAH4 at pH 8 and alamethicin strongly influence the mobility of the glycerol groups. The 20 amino acid peptide alamethicin has a largely linear α-helical structure spanning the membrane with the molecular axis tilted by 10° relative to the bilayer normal in POPC bilayers.42 The 20 residues of this peptide form a hydrophobic assembly with one face of the helix being more polar than the other, and thus a moderately amphipathic structure is obtained. In a helical conformation, the length of the peptide matches well the hydrophobic thickness of the bilayer.43 LAH4 is a 26 amino acid peptide which in micellar environments at pH 7.8 adopts a largely hydrophopic α-helical conformation encompassing 18 residues.18 Four histidines add amphipathic character to this helix, the tilt angle of which was estimated at neutral pH from 2D solid-state NMR spectra to be about 10−15° relative to the membrane normal in POPC bilayers.44 Although the proline residues in alamethicin and the accumulation of charged histidines in LAH4 at reduced pH induce a tendency for kinked structures in both peptides,18,42,45 extended linear conformations have been shown to be stabilized in transmembrane configurations.46 We speculate that polar groups of the α-helical peptides form hydrogen bonds with the lipid acyl groups and thus strongly decrease their rotational and translational mobility.26 These polar groups could be from backbone residues that are exposed (Gly 11 in alamethicin) or not involved in H-bonds (next to Pro 2 and Pro 14 in alamethicin or at the nonstructured termini which are close to the interface), from the very termini of the peptide sequences or from side chains such as His (residues 10, 11, 14, and 18 in LAH4), Lys (two at each terminus of LAH4), or Gln/Glu (residues 7, 18, and 19 of alamethicin). What Can We Learn from the Time-Dependent Fluorescence Shift and Steady-State GP about the Interaction of TMPs with Lipid Molecules? Motivated by the remarkable sensitivity of TDFS reported by Laurdan toward the incorporation of antibacterial peptides with transmembrane orientation, we have investigated the effect of model peptides which mimic transmembrane domains of integral membrane proteins in cells on the mobility of the bilayer acyl groups. LW21 peptide, W2(L)8A(L)8W2, is a representative of model peptides which mimic transmembrane domains of integral membrane proteins in cells and contains a hydrophobic sequence of 17 small amino acids flanked by tryptophan residues. The latter amino acids are known to interact with the membrane interface and thereby act as anchoring sites for transmembrane helices.47,48 In recent studies, it was shown that a peptide similar to WALP23, AW2L(AL)8W2A, fits well into the Ld domain of DOPC bilayers.49 For this system, MD simulations predicted intermediate tilt angles of about 15 to 20°, which mainly result from thermal fluctuations and indicate hydrophobic matching conditions. The hydrophobic thick-

nesses (an important factor for peptide−membrane matching) for POPC and DOPC (27.1 and 26.8 Å, respectively) are very close to each other.50 Similar to alamethicin, a 100% increase in τr due to LW21 insertion is observed for a 10:1 lipid to protein ratio. The polar groups of this model transmembrane helix located below the external interface of the bilayer, i.e., in the region where the tryptophans are expected,51 exhibit a strong influence on the mobility of the POPC acyl groups. Remarkably, the probed polarity (Δν) is increased. As already discussed above, the increased polarity (together with the decreased mobility) suggests a direct interaction between polar residues of the LW21 peptide and POPC acyl groups. As in the case of LAH4 at pH 8 and alamethicin, slowing down of the TDFS kinetics is reflected in an increase in the GP values deduced from Laurdan steady-state spectra. The TDFS data show that the observed blue shift in the steady-state spectrum (expressed as an increase in GP) is due to a lower mobility of the chromophore’s environment and not to a decrease in water penetration. Apparently, in the presence of transmembrane peptides Laurdan GP does not give information on the changes in polarity or hydration, while Δν shows an increase in the probed polarity due to the presence of the TMP. In other words, the increase in polarity here probed should lead to a decrease in GP, while the decrease in the mobility should lead to an increase. As matter of fact, we detect a significant increase in GP. Thus, these experiments suggest that Laurdan’s GP value in the case examined here is more sensitive to mobility than polarity at the glycerol level of lipid bilayers. Considering the fact that a cell membrane is crowded with such transmembrane domains, our results indicate that a more careful interpretation of Laurdan GP values in cell membranes might be needed. Finally, we investigated the LAT peptide derived from the transmembrane domain and proximal sequences of the LAT signaling adaptor (type I protein) present at the plasma membrane of T lymphocytes. As for the other three examined peptides showing a transmembrane localization, an increase in τr due to the presence of LAT peptide is observed. However, the effect is smaller (i.e., about 20% for a 10:1 lipid to peptide ratio). The tilt angle for LAT transmembrane orientation has not been determined. However, that parameter was recently calculated for WALP31, which, according to Schaefer et al.,49 carries a hydrophobic stretch of comparable length as LAT TM (EADWLSPVGLG(L) 4 PFLVTLLAALCVRCRE). For WALP31 in DOPC bilayers, a tilt angle of 45° was predicted by molecular dynamics simulations. Although such a large tilt angle seems to be quite unstable with regard to lipid packing and the LAT peptide examined here cannot be directly compared to WALP31, the amino acid sequence suggests that LAT might show a hydrophobic mismatch in POPC bilayers and thus react by a certain deviation from a “perfect” transmembrane orientation.49 Moreover, the LAT transmembrane domain is a rather complicated sequence with residues such as proline and glycine which can cause breaks in the helical structure of the peptide. Thus, LAT is supposed to be a more flexible and a less linear α-helical peptide compared to alamethicin or LW21. We speculate that LAT, being a more flexible peptide, might to a lesser extent hinder the mobility of the acyl groups of the lipids than LW21. In summary, our experiments demonstrate that the incorporation of TMPs decreases the degree of lipid mobility at the glycerol level, while the probed polarity is unchanged or increased. In particular, the lipid acyl groups experience 6175

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179

Langmuir

Article

lower peptide to lipid ratio is sufficient to induce the leakage of dye from vesicles. It is most likely one of the reasons for the higher antimicrobial activity of LAH4 under acidic conditions reported previously.20,21 FCS Dye Leakage Assay in the Presence of LAH4: Influence of Negatively Charged Lipids on the Permeation Efficiency. To assess the influence of lipid bilayer charge on its permeation by LAH4, we repeated leakage experiments with LUVs composed of anionic lipids (POPG) and a mixture of zwitterionic and anionic lipids (POPC/POPG 7/3). Results in Figure 3 show that the difference between pH 4.5 and pH 8 is less pronounced in the case of negatively charged LUVs. Furthermore, it is evident that higher concentrations of LAH4 are needed to induce the release of entrapped dye from LUVs composed of negatively charged lipids (POPG) than from those composed of zwitterionic lipid (POPC). This is in agreement with Vogt and Bechinger,20 who found that an order of magnitude lower concentration of LAH4 was required for the release of dye from POPC compared to POPG vesicles. It also shows that the difference in surface charge between membranes of prokaryotic and eukaryotic cells is not sufficient to explain the selectivity of LAH4 against bacteria20,21,55 and that other factors (such as the stabilizing role of sterols in eukaryotic membranes)56−58 are involved in the biological activity of LAH4. FCS of Vesicle Suspensions: Does LAH4 Form Pores or Disintegrate Vesicles? To distinguish whether the entrapped dye is released from LUVs via pores in membranes of the vesicles or whether the release is accompanied by the disintegration of LUVs into smaller fragments (which could invalidate the interpretation of the TDFS data), we followed changes in the size and number of LUVs (containing fluorescently labeled lipids) treated with LAH4 by FCS. Suspensions of vesicles of the same lipid compositions, for which dye leakage assay was performed, in buffers of both pH values were titrated with LAH4, and correlation curves for each peptide to lipid ratio were fitted to the model (eq 4). Experiments were repeated, and the average values of diffusion time τD and number of independent particles in the detection volume N for each peptide to lipid ratio were determined. Both τD and N remained constant within experimental error in all cases throughout the whole titration up to the highest peptide to lipid ratios (1:10). That finding clearly shows that the disintegration of vesicles into smaller fragments (which would be manifested by a decrease in τD and an increase in N) does not take place and that the observed leakage happens via openings or pores in the LUV membranes.

hindered mobility due to an interaction with the peptide. The extent of this effect appears to be correlated with intrinsic properties such as the length and flexibility of individual TMPs. FCS Dye Leakage Assay on Neutral LUVs in the Presence of LAH4: Permeation Efficiency of the Transmembrane Versus Peripheral Binding Mode. The TDFS measurements confirmed that in fully hydrated vesicles an increase in the pH triggers the transition of a peripheral to transmembrane localization of LAH4. The next step was to connect the particular membrane location with the membrane permeation efficiency. To this end, suspensions of LUVs (100 μM lipid concentration) loaded with Alexa 488 were treated with increasing concentrations of LAH4, and measured FCS curves were fitted with two models (eqs 4 and 5). The diffusion time τDB of free Alexa 488 was determined in a reference measurement and set to 115 μs. Fractions Fent of Alexa 488 entrapped within the vesicles for each peptide to lipid ratio were calculated using eqs 7 and 8 using data from the whole titration series. The results are displayed in Figure 3. The

Figure 3. FCS dye leakage assay. LUV suspensions (100 μM lipid concentration) of different lipid compositions (POPC − squares, POPC/POPG = 7/3 − triangles, POPG − circles) with entrapped Alexa 488 were titrated with LAH4 dissolved in the same buffer. The fraction of entrapped dye for each peptide to lipid ratio was normalized to the initial fraction before the addition of peptide. Acidic (pH 4.5, closed symbols) and basic (pH 8, open symbols) conditions are compared. Average values from repeated experiments and their standard deviations are shown.

fractions of entrapped dye have been normalized to the fraction before peptide addition, which was set to 1. In reality, the initial ratio Fent(0) before the first addition of peptide was already lower than 1, with the average value being 0.65. That was most likely caused by the imperfect separation of free Alexa 488 during dialysis as well as the formation of a supported lipid bilayer on the walls of the measurement chamber. In the latter process, a certain fraction of vesicles adsorb to the walls and burst, releasing the entrapped dye.52,53 Differences in Fent(0) between individual samples were probably caused by the propensities of SLB formation as a function of lipid composition.54 Therefore, the fractions Fent were normalized to make the comparison of their dependencies on the peptide to lipid ratio for various lipid compositions easier. First we investigated the influence of pH (and thus peptide orientation) on the ability of LAH4 to permeate zwitterionic (POPC) membranes. As shown in Figure 3, permeation is more efficient at acidic pH, when peptide helices are oriented parallel to the membrane surface. Under those conditions, a

■

CONCLUSIONS AND OUTLOOK This article addresses two particular issues: (1) The TDFS method is introduced to identify the mode of peptide orientation in membranes and to characterize the effect of TMPs on the bilayer organization. (2) The efficiency and mechanism of pH-dependent membrane penetration of LAH4 has been investigated in more detail, and thus important information on its mode of membrane interaction has been obtained. In (1), TDFS sensed by Laurdan in POPC bilayers in the presence of TMPs (LAH4 at basic pH, alamethicin, LW21, and LAT) indicates that there is a direct interaction between the peptide and acyl groups of the bilayer. This interpretation is based on the observation that the incorporation of peptide strongly hinders the mobility of lipid acyl groups but does not change or even increase the polarity probed by Laurdan. 6176

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179

Langmuir

Article

(2) Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415, 389−395. (3) Castro, M. S.; Cilli, E. M.; Fontes, W. Combinatorial synthesis and directed evolution applied to the production of alpha-helix forming antimicrobial peptides analogues. Curr. Protein Pept. Sci. 2006, 7, 473−478. (4) Marr, A. K.; Gooderham, W. J.; Hancock, R. E. W. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr. Opin. Pharmacol. 2006, 6, 468−472. (5) Matsuzaki, K.; Sugishita, K.; Harada, M.; Fujii, N.; Miyajima, K. Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria. Biochim. Biophys. Acta, Biomembr. 1997, 1327, 119−130. (6) Sato, H.; Feix, J. B. Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic alpha-helical antimicrobial peptides. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 1245−1256. (7) Bechinger, B. The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy. Biochim. Biophys. Acta, Biomembr. 1999, 1462, 157−183. (8) Shai, Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta, Biomembr. 1999, 1462, 55−70. (9) Matsuzaki, K. Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta, Biomembr. 1999, 1462, 1−10. (10) Hancock, R. E. W.; Scott, M. G. The role of antimicrobial peptides in animal defenses. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8856−8861. (11) Dathe, M.; Nikolenko, H.; Meyer, J.; Beyermann, M.; Bienert, M. Optimization of the antimicrobial activity of magainin peptides by modification of charge. FEBS Lett. 2001, 501, 146−150. (12) Porter, E. A.; Weisblum, B.; Gellman, S. H. Mimicry of hostdefense peptides by unnatural oligomers: Antimicrobial beta-peptides. J. Am. Chem. Soc. 2002, 124, 7324−7330. (13) Oh, J. E.; Hong, S. Y.; Lee, K. H. Design, synthesis and characterization of antimicrobial pseudopeptides corresponding to membrane-active peptide. J. Pept. Res. 1999, 54, 129−136. (14) Aisenbrey, C.; Kinder, R.; Goormaghtigh, E.; Ruysschaert, J. M.; Bechinger, B. Interactions involved in the realignment of membraneassociated helices - An investigation using oriented solid-state NMR and attenuated total reflection Fourier transform infrared spectroscopies. J. Biol. Chem. 2006, 281, 7708−7716. (15) Bechinger, B. Towards membrane protein design: pH-sensitive topology of histidine-containing polypeptides. J. Mol. Biol. 1996, 263, 768−775. (16) Sheynis, T.; Sykora, J.; Benda, A.; Kolusheva, S.; Hof, M.; Jelinek, R. Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies. Eur. J. Biochem. 2003, 270, 4478−4487. (17) Zhang, Y. P.; Lewis, R.; Henry, G. D.; Sykes, B. D.; Hodges, R. S.; McElhaney, R. N. Peptide Models of Helical Hydrophobic Transmembrane Segments of Membrane Proteins. 1. Studies of the Conformation, Intrabilayer Orientation, and Amide Hydrogen Exchangeability of Ac-K2-(LA)12-K2-Amide. Biochemistry 1995, 34, 2348−2361. (18) Georgescu, J.; Munhoz, V. H. O.; Bechinger, B. NMR Structures of the Histidine-Rich Peptide LAH4 in Micellar Environments: Membrane Insertion, pH-Dependent Mode of Antimicrobial Action, and DNA Transfection. Biophys. J. 2010, 99, 2507−2515. (19) Bechinger, B.; Ruysschaert, J. M.; Goormaghtigh, E. Membrane helix orientation from linear dichroism of infrared attenuated total reflection spectra. Biophys. J. 1999, 76, 552−563. (20) Vogt, T. C. B.; Bechinger, B. The interactions of histidinecontaining amphipathic helical peptide antibiotics with lipid bilayers The effects of charges and pH. J. Biol. Chem. 1999, 274, 29115−29121.

Analogous experiments with peptides known to adopt a membrane orientation parallel to the surface (magainin 2 and LAH4 at acidic pH) show no changes in Laurdan TDFS (GP changes are also within the experimental error). The experiments demonstrate that TDFS can be a useful tool for the characterization of the orientations of membrane-associated peptides. When comparing the two model peptides mimicking transmembrane domains of integral membrane proteins, it appears that the magnitude of TDFS sensed by Laurdan is larger for a linear α-helical structure oriented parallel to the lipid molecules (LW21) compared to peptide with a higher probability of accommodating a more flexible structure (LAT). The impact of the hydrophobic mismatch on the properties of lipid bilayers has been the focus of interest,48,59 and we plan to study the impact of an analogous model and TMD-derived peptides of varying length and flanking residues on TDFS in lipid bilayers. Due to the robustness and sensitivity of the TDFS method,26 we believe that we better understand some of the molecular details that are associated with hydrophobic mismatch conditions and can identify the functional groups directly interacting with the acyl groups of the bilayer. In this context, it should be mentioned that the TDFS method was recently also employed on cells.60 In (2), membrane permeation induced by LAH4 was studied by an FCS dye leakage assay, which showed that the peptide in an in-plane orientation permeates membranes more efficiently and that the permeation of zwitterionic membranes is more efficient compared to that of negatively charged ones. FCS of vesicle suspensions demonstrated that LAH4 does not disintegrate vesicles and that the permeation, therefore, happens most likely via the formation of pores or small openings.

■

AUTHOR INFORMATION

Corresponding Author

*Telephone: (+420) 266053264. E-mail: martin.hof@jh-inst. cas.cz. Present Address

R.M.: Centre for BioImaging Sciences, Departments of Biological Sciences and Chemistry, National University of Singapore, 14 Science Drive 4, Singapore 117546. Author Contributions

R.M. and P.J. contributed equally to this work. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Support from the Czech Science Foundation via grant P208/ 12/G016 is acknowledged. Moreover, M.H acknowledges the Praemium Academie Award (Academy of Sciences of the Czech Republic). The Bechinger team acknowledges the financial contributions of the Agence Nationale de la Recherche (projects TRANSPEP and the LabEx Chemistry of Complex Systems), the University of Strasbourg, the CNRS, and the RTRA International Center of Frontier Research in Chemistry. M.C. acknowledges the Purkyne Fellowship of the Academy of Sciences of the Czech Republic.

■

REFERENCES

(1) Hadley, E. B.; Hancock, R. E. W. Strategies for the Discovery and Advancement of Novel Cationic Antimicrobial Peptides. Curr. Top. Med. Chem. 2010, 10, 1872−1881. 6177

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179

Langmuir

Article

Fluorescence Spectroscopy and Molecular Simulations. Langmuir 2010, 26, 6140−6144. (41) Jurkiewicz, P.; Cwiklik, L.; Vojtiskova, A.; Jungwirth, P.; Hof, M. Structure, dynamics, and hydration of POPC/POPS bilayers suspended in NaCl, KCl, and CsCl solutions. Biochim. Biophys. Acta, Biomembr. 2012, 1818, 609−616. (42) Salnikov, E. S.; Friedrich, H.; Li, X.; Bertani, P.; Reissmann, S.; Hertweck, C.; O’Neil, J. D. J.; Raap, J.; Bechinger, B. Structure and Alignment of the Membrane-Associated Peptaibols Ampullosporin A and Alamethicin by Oriented N-15 and P-31 Solid-State NMR Spectroscopy. Biophys. J. 2009, 96, 86−100. (43) Kessel, A.; Schulten, K.; Ben-Tal, N. Calculations suggest a pathway for the transverse diffusion of a hydrophobic peptide across a lipid bilayer. Biophys. J. 2000, 79, 2322−2330. (44) Vidovic, V. Structural Investigations of Peptide-DNA Transfection Complexes: Bacterial Overexpression and Isotopic Labelling of Peptides, Biophysical and NMR Investigations. Ph.D. Thesis, University of Strasbourg, Strasbourg, France, 2011. (45) Sansom, M. S. P. Alamethicin and related peptaibols - Model ion channels. Eur. Biophys. J. Biophys. Lett. 1993, 22, 105−124. (46) Page, R. C.; Kim, S.; Cross, T. A. Transmembrane helix uniformity examined by spectral mapping of torsion angles. Structure 2008, 16, 787−797. (47) Wallin, E.; Tsukihara, T.; Yoshikawa, S.; VonHeijne, G.; Elofsson, A. Architecture of helix bundle membrane proteins: An analysis of cytochrome c oxidase from bovine mitochondria. Protein Sci. 1997, 6, 808−815. (48) Holt, A.; Killian, J. A. Orientation and dynamics of transmembrane peptides: the power of simple models. Eur. Biophys. J. Biophys. Lett. 2010, 39, 609−621. (49) Schafer, L. V.; de Jong, D. H.; Holt, A.; Rzepiela, A. J.; de Vries, A. H.; Poolman, B.; Killian, J. A.; Marrink, S. J. Lipid packing drives the segregation of transmembrane helices into disordered lipid domains in model membranes. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 1343−1348. (50) Kucerka, N.; Tristram-Nagle, S.; Nagle, J. F. Structure of fully hydrated fluid phase lipid bilayers with monounsaturated chains. J. Membr. Biol. 2005, 208, 193−202. (51) de Planque, M. R. R.; Kruijtzer, J. A. W.; Liskamp, R. M. J.; Marsh, D.; Greathouse, D. V.; Koeppe, R. E.; de Kruijff, B.; Killian, J. A. Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane alpha-helical peptides. J. Biol. Chem. 1999, 274, 20839−20846. (52) Richter, R. P.; Berat, R.; Brisson, A. R. Formation of solidsupported lipid bilayers: An integrated view. Langmuir 2006, 22, 3497−3505. (53) Weirich, K. L.; Israelachvili, J. N.; Fygenson, D. K. Bilayer Edges Catalyze Supported Lipid Bilayer Formation. Biophys. J. 2010, 98, 85− 92. (54) Wacklin, H. P.; Thomas, R. K. Spontaneous formation of asymmetric lipid bilayers by adsorption of vesicles. Langmuir 2007, 23, 7644−7651. (55) Mason, A. J.; Moussaoui, W.; Abdelrahman, T.; Boukhari, A.; Bertani, P.; Marquette, A.; Shooshtarizaheh, P.; Moulay, G.; Boehm, N.; Guerold, B.; Sawers, R. J. H.; Kichler, A.; Metz-Boutigue, M. H.; Candolfi, E.; Prevost, G.; Bechinger, B. Structural Determinants of Antimicrobial and Antiplasmodial Activity and Selectivity in Histidinerich Amphipathic Cationic Peptides. J. Biol. Chem. 2009, 284, 119− 133. (56) Stromstedt, A. A.; Ringstad, L.; Schmidtchen, A.; Malmsten, M. Interaction between amphiphilic peptides and phospholipid membranes. Curr. Opin. Colloid Interface Sci. 2010, 15, 467−478. (57) Taheri-Araghi, S.; Ha, B. Y. Cationic antimicrobial peptides: a physical basis for their selective membrane-disrupting activity. Soft Matter 2010, 6, 1933−1940. (58) Sood, R.; Kinnunen, P. K. J. Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L. Biochim. Biophys. Acta, Biomembr. 2008, 1778, 1460− 1466.

(21) Mason, A. J.; Gasnier, C.; Kichler, A.; Prevost, G.; Aunis, D.; Metz-Boutigue, M. H.; Bechinger, B. Enhanced membrane disruption and antibiotic action against pathogenic bacteria by designed histidinerich peptides at acidic pH. Antimicrob. Agents Chemother. 2006, 50, 3305−3311. (22) Langlet-Bertin, B.; Leborgne, C.; Scherman, D.; Bechinger, B.; Mason, A. J.; Kichler, A. Design and Evaluation of Histidine-Rich Amphipathic Peptides for siRNA Delivery. Pharm. Res. 2010, 27, 1426−1436. (23) Zhang, T. T.; Kang, T. H.; Ma, B.; Xu, Y. J.; Hung, C. F.; Wu, T. C. LAH4 enhances CD8+T cell immunity of protein/peptide-based vaccines. Vaccine 2012, 30, 784−793. (24) Fenard, D.; Genries, S.; Scherman, D.; Galy, A.; Martin, S.; Kichler, A. Infectivity enhancement of different HIV-1-based lentiviral pseudotypes in presence of the cationic amphipathic peptide LAH4L1. J. Virol. Methods 2013, 189, 375−378. (25) Demchenko, A. P.; Mely, Y.; Duportail, G.; Klymchenko, A. S. Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes. Biophys. J. 2009, 96, 3461−3470. (26) Jurkiewicz, P.; Cwiklik, L.; Jungwirth, P.; Hof, M. Lipid hydration and mobility: An interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations. Biochimie 2012, 94, 26−32. (27) Kaiser, H. J.; Orlowski, A.; Rog, T.; Nyholm, T. K. M.; Chai, W. G.; Feizi, T.; Lingwood, D.; Vattulainen, I.; Simons, K. Lateral sorting in model membranes by cholesterol-mediated hydrophobic matching. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16628−16633. (28) Shogomori, H.; Hammond, A. T.; Ostermeyer-Fay, A. G.; Barr, D. J.; Feigenson, G. W.; London, E.; Brown, D. A. Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells. J. Biol. Chem. 2005, 280, 18931−18942. (29) Parasassi, T.; De Stasio, G.; Dubaldo, A.; Gratton, E. Phase Fluctuation in Phospholipid-Membranes Revealed by Laurdan Fluorescence. Biophys. J. 1990, 57, 1179−1186. (30) Horng, M. L.; Gardecki, J. A.; Papazyan, A.; Maroncelli, M. Subpicosecond Measurements of Polar Solvation Dynamics Coumarin-153 Revisited. J. Phys. Chem. 1995, 99, 17311−17337. (31) Gregor, I.; Patra, D.; Enderlein, J. Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation. ChemPhysChem 2005, 6, 164−170. (32) Benda, A.; Benes, M.; Marecek, V.; Lhotsky, A.; Hermens, W. T.; Hof, M. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir 2003, 19, 4120−4126. (33) Elson, E.; Magde, D. Fluorescence Correlation Spectroscopy. I. Conceptual Basis and Theory. Biopolymers 1974, 13, 1−27. (34) Magde, D.; Elson, E. Fluorescence Correlation Spectroscopy. II.An Experimental Realization. Biopolymers 1974, 13, 29−61. (35) Widengren, J.; Mets, U.; Rigler, R. Fluorescence Correlation Spectroscopy of Triplet-States in Solution - a Theoretical and Experimental-Study. J. Phys. Chem. 1995, 99, 13368−13379. (36) Yu, L.; Ding, J. L.; Ho, B.; Wohland, T. Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: An amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and antimicrobial activity. Biochim. Biophys. Acta, Biomembr. 2005, 1716, 29−39. (37) Pramanik, A.; Thyberg, P.; Rigler, R. Molecular interactions of peptide with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy. Chem. Phys. Lipids 2000, 104, 35−47. (38) Yu, L. L.; Guo, L.; Ding, J. L.; Ho, B.; Feng, S. S.; Popplewell, J.; Swann, M.; Wohland, T. Interaction of an artificial antimicrobial peptide with lipid membranes. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 333−344. (39) Sanchez, S. A.; Tricerri, M. A.; Gratton, E. Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7314−7319. (40) Beranova, L.; Cwiklik, L.; Jurkiewicz, P.; Hof, M.; Jungwirth, P. Oxidation Changes Physical Properties of Phospholipid Bilayers: 6178

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179

Langmuir

Article

(59) Ramadurai, S.; Holt, A.; Schafer, L. V.; Krasnikov, V. V.; Rijkers, D. T. S.; Marrink, S. J.; Killian, J. A.; Poolman, B. Influence of Hydrophobic Mismatch and Amino Acid Composition on the Lateral Diffusion of Transmembrane Peptides. Biophys. J. 2010, 99, 1447− 1454. (60) Ghosh, S.; Chattoraj, S.; Mondal, T.; Bhattacharyya, K. Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell: Time-Resolved Confocal Microscopy. Langmuir 2013, 29, 7975− 7982.

6179

dx.doi.org/10.1021/la5006314 | Langmuir 2014, 30, 6171−6179