a helix-induced oligomeric transition of gaegurin 4, an
TRANSCRIPT
Mol. Cells, Vol. 21, No. 2, pp. 229-236
A Helix-induced Oligomeric Transition of Gaegurin 4, an Antimicrobial Peptide Isolated from a Korean Frog
Su-Yong Eun1, Hae-Kyung Jang
2, Seong-Kyu Han
3,†, Pan-Dong Ryu
3, Byeong-Jae Lee
4, Kyou-Hoon Han
5,
and Soon-Jong Kim2,
* 1 Department of Physiology, College of Medicine, Cheju National University, Jeju 690-756, Korea; 2 Department of Chemistry, Mokpo National University, Chonnam 534-729, Korea; 3 College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea; 4 Laboratory of Molecular Genetics, Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National
University, Seoul 151-742, Korea; 5 Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea.
(Received November 1, 2005; Accepted January 10, 2006)
Gaegurin 4 (GGN4), a novel peptide isolated from the skin of a Korean frog, Rana rugosa, has broad spec-trum antimicrobial activity. A number of amphipathic peptides closely related to GGN4 undergo a coil to he-lix transition with concomitant oligomerization in lipid membranes or membrane-mimicking environments. Despite intensive study of their secondary structures, the oligomeric states of the peptides before and after the transition are not well understood. To clarify the structural basis of its antibiotic action, we used ana-lytical ultracentrifugation to define the aggregation state of GGN4 in water, ethyl alcohol, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The maximum size of GGN4 in 15% HFIP corresponded to a decamer, whereas it was monomeric in buffer. The oligomeric transition is accompanied by a cooperative 9 nm blue-shift of maximum fluorescence emission and a large secondary structure change from an almost random coil to an α-helical structure. GGN4 induces pores in lipid membranes and, using electrophysiological meth-ods, we estimated the diameter of the pores to be exceed 7.3 Å, which suggests that the minimal oligomer struc-ture responsible is a pentamer. Keywords: Aggregation State; Analytical Ultracentri-
fuge; Antimicrobial Peptide; Gaegurin 4.
† Present address: School of Dentistry, Chonbuk National Uni-
versity, Jeonju 561-756, Korea.
* To whom correspondence should be addressed.
Tel: 82-61-450-2338; Fax: 82-61-450-2339
E-mail: [email protected]
Introduction
In the last two decades, more than 500 antimicrobial pep-
tides (AMPs) have been identified in host defense systems
from insects to animals (Boman, 1995; Rinaldi, 2002;
Zasloff, 2002). AMPs are an essential part of innate immu-
nity for combating microbial challenges. These small pep-
tides are multifunctional effectors of immunity on skin and
mucosal surfaces and have antimicrobial activity against
various bacteria, viruses, fungi, cancer cells and parasites.
The skin secretions of frogs contain many different types of
AMPs (Rinaldi, 2002; Zasloff, 2002). The gaegurins are six
antimicrobial peptides, isolated from the frog, Rana rugosa,
that can be classified into two families based on their lengths
and sequence similarities (Park et al., 1994). Gaegurin 4
(GGN4) belongs to gaegurin family I and has activity
against Gram negative and Gram-positive bacteria, fungi
and protozoa (Park et al., 1994). Recently, a gaegurin fam-
ily II peptide (gaegurin 6, GGN6) was reported to also have
antitumor activity (Kim et al., 2003).
GGN4 (GILDTLKQFAKGVGKDLVKGAAQGVLST-
VSCKLAKTC) consists of 37 amino acid residues with 4
net positive charges (6 K & 2 D) and a number of hydro-
phobic residues (Park et al., 1994). Like brevinins,
ranalexins, and esculentins, all antibiotic peptides from
the Rana genus, GGN4 contains a well-conserved disul-
fide bridge between C31 and C37 (CKLAKTC), termed a
Rana box. The Rana box (heptapeptide motif) seems to be
dispensable for antimicrobial (Park et al., 2000) and anti-
tumor (Kim et al., 2003) activities, but is required for
good pore-forming activity in artificial lipid membranes
Abbreviations: GGN4, gaegurin 4; HFIP, hexafluoro-2-propanol.
Molecules
and
Cells©KSMCB 2006
230 Oligomeric State of Gaegurin 4
(Kim et al., 1999). Truncation of GGN4 beyond the
hetapeptide form causes noticeable decreases in antimicro-
bial activity (Kim et al., 1999a) and membrane conduc-
tance measured by a patch-clamp technique (Kim et al.,
1999b).
In many proteins or peptides, hetero-association and
self-association of protein monomers via multimerization
interfaces, such as α-helical structures, to form higher
order oligomers play important roles in biological activity
(Marianayagam et al., 2004; Stewart, 1993). GGN4 con-
tains two amphipathic α-helices (Park et al., 2000) (resi-
dues 2 to 10 and 16 to 32, underlined) and has been
shown to form cation-selective pores in lipid membranes
(Kim et al., 1999a; 2004). Despite the pore forming ac-
tion and the presence of helices, only monomers were
reported in the conditions for NMR analysis (Park et al.,
2000; 2002; Suh et al., 1996). However, a recent NMR
study of GGN5 revealed that some hydrophobic residues
(Leu-5, Phe-6, Val-8, Ala-8, Ala-9, Val-12 and Val-16)
underwent slow proton exchange (Park et al., 2002). Such
slow exchange was also observed for some residues (Ile-
17 and Ile-20) of an even shorter gaegurin, GGN6 (Suh et
al., 1996), and suggested the possible existence of mul-
timerization interfaces in gaegurins. Alamethicin (He et al.,
1996), protegrin (Yang et al., 2000) magainins (Matsu-
zaki et al., 1995; 1996; Yang et al., 2000) melittins (Yang
et al., 2001) and cecropins (Mchaourab et al., 1993) are
well-known peptide antibiotics that form amphipathic heli-
cal configurations and higher- order oligomeric structures.
Even though oligomerization is important for the func-
tion of many biological macromolecules, including antim-
icrobial peptides, direct proof of aggregation and deter-
mination of the number of molecules in the aggregate
have been difficult to achieve due to the lack of suitable
high-resolution methods that can detect the higher-order
structures formed by these peptides in membrane envi-
ronments. In this study, we have used analytical ultracen-
trifugation to estimate the size of GGN4 in 1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP) [which promotes α-helix
formation in amphipathic peptides (Hirota et al., 1997)],
and patch-clamp techniques to estimate the minimal pore
size in artificial membranes. We also used steady-state
fluorescence to follow microenvironmental changes near
an introduced fluorescence probe and a CD technique to
monitor overall secondary structural changes.
Materials and Methods
Peptide sample The natural GGN4 peptide was purified from
the skin of Rana rugosa as described previously (Park et al.,
1994). The purity of the peptide was greater than 99%, as con-
firmed by mass-spectroscopy and analytical HPLC. HFIP was
obtained from Sigma (USA) and fluorescein 5-isothiocyanate
(FITC) from Molecular Probes (Eugene, USA). Synthetic phos-
pholiplids such as palmitoyl-oleoyl-phosphatidylethanolamine
(PE), palmitoyl-oleoyl-phosphatidylcho-line (PC), and palmi-
toyl-oleoyl-phosphatidylserine (PS) were purchased from Avanti
Polar Lipids (Alabaster, USA). All other reagents were of the
highest analytical grade from Sigma. The extinction coefficient of
full-length GGN4 at 220 nm was estimated to be 30076 M-1cm-1
from its UV spectrum and peptide mass. The latter was deter-
mined by the Harvard Microchemical Core Facility.
Equilibrium sedimentation Equilibrium sedimentation studies
were performed using a Beckman ProteomeLab XL-A analytical
ultracentrifuge at the Analytical Ultracentrifuge Core Facility,
Mokpo National University. A GGN4 sample in the absence of
HFIP was measured at 25°C using a 4-hole rotor with a counter-
balance and aluminum centerpiece cells, at two speeds, 40,000
rpm and 50,000 rpm, in 2.5 mM phosphate buffer containing 1
mM EDTA, at pH 3.3 or in 1× PBS buffer at pH 7.4. The data at
the two speeds were fitted to the appropriate models. Experi-
ments in the presence of HFIP were performed with the 4-hole
rotor at 30,000 revs/min in the same two buffers. The peptide
concentration was 25 µM (0.1 mg/ml). The time required for the
attainment of equilibrium was established by running at the
given rotor speed until scans were consistent over 12 h; this was
achieved by at most 36 h. The distribution of the samples within
the cells was determined by measuring absorbance at 220 nm or
230 nm. The samples were also scanned at 330 nm, where the
peptide has no absorption, to obtain a baseline. Five scans were
collected and averaged to give the final averaged data. The par-
tial specific volume of GGN4 at 25°C was calculated to be
0.7487, from the partial specific volume of its constituent amino
acids. A calculated molecular mass of 3734.7 Daltons was used
for the data analysis.
For mathematical modeling by non-linear least-squares curve
fitting, the fitting function was:
Cr = Cb exp(ApMp(r2-rb
2)) + ε (1)
AP = (1-υρ )ω2/RT
where Cr is the total concentration at radial position r, Cb is the
concentration of peptide at the bottom of the cell, Mp is the mo-
lecular weight of the peptide, υ is the partial specific volume
and ρ is the solution density, ω is the rotor angular velocity,
and ε is a baseline error term. The model was selected by
examining the weighted sum of square values and weighted root
mean square error values. Further data manipulation and data
analysis were performed using MLAB (Knott, 1979) with the
data analysis computer.
Circular dichroism spectra CD spectroscopy was carried out
on a JASCO J-715s in Mokpo National University Central
Laboratory with a 1 mm cell at 0.1 mg/ml concentration at 25°C
in 2.5 mM potassium phosphate buffer, pH 3.3, or other condi-
tions as indicated. Five accumulated scans were taken for each
sample at a speed of 50 nm/min. The temperature was controlled
by a Neslab RT111 circulating water bath.
Su-Yong Eun et al. 231
Electrochemical measurements and data analysis. Planar
lipid bilayers Solvent-containing lipid bilayers were formed
across the tip of a patch pipette (Coronado and Latorre, 1983;
Cruciani et al., 1992), and in neutral tip-dip bilayers the perme-
ability ratio of K to Cl in GGN4-induced conductances was
similar to that in painted bilayers (Kim et al., 1999a). Patch
pipettes were pulled with a two-stage puller (PP-83, Narishige
Scientific Instr., Tokyo, Japan) and filled with a pipette solution
containing 10 mM HEPES-KOH and 100 mM KCl (pH 7.2).
First, a patch pipette was lowered below the solution level in the
recording chamber (1.2 ml) filled with the pipette solution, and
a drop of the lipid solution composed of phosphatidylethanola-
mine: phosphatidylcholine: cholesterol (72:18:10), in n-decane
(10 mg/ml), was layered over the pipette-water interface. Then
the pipette was carefully withdrawn into the lipid drop and low-
ered again into the bath solution. The formation and size of the
bilayer was estimated by monitoring the membrane capacitances
as described for the painted bilayer. The membrane capacitance
of the tip-dip bilayers was approximately 15 pF. GGN4 was
added to the bath before perfusion and the bath was perfused
(0.7 ml/min) with test solutions (12 ml) after channel activity
was detected.
Electrical measurements and data analysis GGN4-induced
conductances were obtained from the slope of the current-
voltage data obtained by a ramp voltage command with a volt-
age clamp amplifier (CEZ2300, Nihonkoden, Japan). The ampli-
fier headstage was connected to the bath via an agar (3%) bridge
to reduce the junction potentials. Electrode asymmetry was cor-
rected in symmetrical salt conditions before formation of the
bilayer. Junction potentials produced by the differences in ion
species were measured at the end of each experiment and sub-
tracted from the command voltage (Cruciani et al., 1992). The
largest junction potential (13−17 mV) occurred between KCl
and LiCl (200 mM). We designated the current flow from bath
to pipette as “outward current”. Current-voltage relations were
obtained by a ramp voltage command from -150 to +150 mV, or
vice versa, for 3 s. Relative permeabilities of monovalent
cations were calculated from the measured reversal potentials
according to the Goldman-Hodgkin- Katz (GHK) equation.
(Coronado and Latorre, 1983; Cruciani et al., 1992) GGN4-
induced currents were stored on a PC using a Labmaster DMA
(TL-1 125 kHz, Axon Inst. Co., USA), VTR tapes with a VR-10
digital data recorder (Instrutech Corp., USA), and a pen recorder.
pClamp software (Ver. 6.03, Axon Inst. Co., USA) for electro-
physiological experiments and data analysis was used for volt-
age commands, measurements of current amplitude and reversal
potentials, and for data illustration.
Fluorescence measurements Due to the absence of tryptophan
or tyrosine in native GGN4 we introduced an extrinsic fluoro-
phore, fluorescein, using fluorescein 5-isothiocyanate (FITC).
Labeling was carried out at pH 7.0 with a 5:1 molar ratio of dye
to peptide to label the N-terminus. Free dye was removed by
gel-filtration on a G-25 column (Pharmacia, Uppsala, Sweden)
followed by dialysis. The labeling ratio was estimated using the
extinction coefficients of the dye (72,000 M−1cm−1 at 494 nm)
and the peptide (30,076 M−1cm−1 at 220 nm). We estimated that
approximately 30% of the GGN4 molecules were labeled. For
fluorescence emission spectra, a Hitachi F-4500 spectro-
fluorimeter was used.
Results
The α-helical propensity of GGN4 The solution confor-
mation of native GGN4 in buffer and various alcohols
was measured by CD (Fig. 1A). In 2.5 mM phosphate
buffer, pH 3.3, the peptide existed as a random coil char-
acterized by a typical minimum at 197 nm. Upon addition
of alcohols, the helicity increased, as evidenced by the
presence of double minima at 208 and 222 nm, and a
positive band at 196 nm. However, the relative intensities
of the mean residue ellipticity at 222 nm were in the or-
der: methanol (MeOH) < ethanol (EtOH) < HFIP in these
conditions. A similar trend was observed in a CD analysis
on melittin (Hirota et al., 1997).
Figure 1B shows the CD spectra of GGN4 at different
HFIP concentrations from 0 to 15% (vol/vol). The peptide
conformation started to transform into a helix at approxi-
mately 6% HFIP and completed its transition near 15%
HFIP. Further addition of HFIP did not cause any notice-
able change. The α-helical content of the native GGN4 in
15% HFIP was estimated to be 58%, using the intensity of
the minimum band at 222 nm (Scholtz et al., 1991).
Along with the changes in the secondary structure, HFIP
also changed the ellipticity ratio (θ222 nm /θ209 nm) from
~0.8 in the absence of HFIP to ~ 1.0 in the presence of
HFIP. This ratio is frequently used as an indication of the
inter-helical interactions yielding oligomeric structures
(Lau et al., 1984).
Oligomeric state of GGN4 in buffer, EtOH and HFIP
The oligomeric state of GGN4 was investigated by ana-
lytical ultracentrifugation, which has been used frequently
to study the oligomeric state of peptides and proteins (Le-
bowitz et al., 2002). Equilibrium sedimentation was car-
ried out in three different conditions: buffer alone, 50%
EtOH, and 15% HFIP. Figure 2 demonstrates the joint fit
(solid line) for the data for GGN4 in 2.5 mM phosphate
buffer, pH 3.3, containing 0.1 mM EDTA at ultracentri-
fuge speeds of 40,000 and 50,000 rpm at 230 nm, assum-
ing homogeneous monomers. For the analysis we used a
monomeric molecular mass of 3,734.5 Da. The root-
mean-square errors of the monomeric joint fit analysis at
the two speeds were approximately 3.50 × 10−3, which
demonstrates the accuracy of the fit. The accuracy of
these fits to the model used was such that it was appropri-
ate to conclude that GGN4 exists as a monomer in buffer,
and that thermodynamic ideality of the solutes had been
232 Oligomeric State of Gaegurin 4
A
B
Fig. 1. CD spectra of GGN4 in various conditions. A. CD spec-
tra of GGN4 in the presence and absence of 15% HFIP, 50%
MtOH and 50% EtOH in 2.5 mM potassium phosphate buffer,
pH 3.3. The concentration of GGN4 was 0.1 mg/ml. B. CD
spectra of GGN4 in the presence of increasing amounts of HFIP
in the same conditions.
established. The actual fit is shown in the lower section,
and a plot of the distribution of the residuals is shown in
the upper section. GGN4 was also examined at another
pH (1× PBS, pH 7.4) and again found to be a monomer
(data not shown). The ultracentrifuge data in 15% HFIP
are presented in Fig. 3. The presence of 15% HFIP in-
duced significant changes in the concentration distribu-
tion of the peptide in the cell, which suggested the pres-
ence of higher order aggregates. Using the appropriate
mathematical models, we determined that the actual dis-
tribution of GGN4 in 15% HFIP was best fitted to a ho-
mogeneous decamer (10-mer, 10×) (Fig. 3). The distribu-
tion of the data was close to the theoretical 10-mer line
and deviated more from 9-mer or 11-mer models. Other
homogeneous (1×, 2×, 4×, 8×, 12×) or interactive (1×↔
2×, 1×↔4×, 1×↔6×, 1×↔10×, 1×↔12×, 1×↔2×↔4×↔
8×....) models were also tested, but they gave inferior re-
sults and were therefore discarded. In order to determine
if other alcohols cause the oligomeric transition, we con-
ducted equilibrium sedimentation in 50% EtOH. Figure 4
shows the effect of 50% EtOH on the oligomeric state of
GGN4 measured at the two different wavelengths (220
Fig. 2. Equilibrium sedimentation analysis of GGN4 in buffer.
The distributions of the absorbance (lower graph) of GGN4 at
equilibrium at 40,000 revs/min (circles) and 50,000 revs/min
(squares) at 25°C in 2.5 mM potassium phosphate buffer, pH 3.3.
The distributions of the residuals (upper graph) as functions of
radial position are also shown. The lines are for a thermody-
namically ideal monomer fitted simultaneously to the data at the
two speeds.
and 230 nm). Unlike HFIP, EtOH failed to cause the pep-
tide to self-associate despite the increased helical content
of GGN4 in its presence (Fig. 1A). Further increase of the
percentage EtOH or MeOH to 70% had no effect (data not
shown).
Fluorinated alcohols, such as 2,2,2-trifluoroethanol (TFE)
and HFIP are known to induce the formation of α-helices
in peptides and proteins. HFIP seems to be the strongest
enhancer of α-helix formation (Kahn et al., 2000; Kuma-
ran and Roy, 1999). In a melittin study, HFIP was found
to be approximately 20 times more effective than ethanol
in promoting helix formation (Hirota et al., 1997). A re-
cent physicochemical analysis indicated that an HFIP-water
binary solution formed micelle-like aggregates of maxi-
mum size at approximately 30% (v/v) HFIP or χHFIP =
0.0671 (Hong et al., 1999; Yoshida et al., 2003). The tet-
rahedral-like water structures that predominate at 0%
HFIP, began to be disrupted to form higher aggregates
upon the addition of HFIP. Even though our experimental
set-up of 15% (v/v) HFIP (χHFIP = 0.0336) is located at
the transition of water to maximal micelle-like aggregates
(Yoshida et al., 2003), it is possible that binding of water-
HFIP co-solvents to GGN4 led us to overestimate GGN4
size (10-mer). However, the decameric GGN4 determined
by equilibrium sedimentation reflects the maximum pos-
sible size of the oligomers in 15% HFIP if no co-solvation
occurred. Until now, only HFIP-water systems have been
Su-Yong Eun et al. 233
Fig. 3. Equilibrium sedimentation analysis of GGN4 in 15%
HFIP. The distributions of the absorbance (lower graph) of
the GGN4 peptide in 2.5 mM potassium phosphate buffer, pH
3.3 containing 15% HFIP at ultracentrifugal equilibrium at
30,000 revs/min and 25°C. The fitted solid line is for a thermo-
dynamically ideal homogeneous 10-mer. The GGN4 monomer
is 3,749.4 Da, and the concentration of GGN4 was 0.1 mg/ml.
The distributions of the residuals (upper graph) fitted to mono-
mers and 10-mers are shown as functions of the radial position.
studied experimentally (Hong et al., 1999; Yoshida et al., 2003); and the effects of HFIP on real peptides have been examined only by molecular dynamic simulations applied to melittin (Roccatano et al., 2005). Fluorescence properties of fluorescein-labeled GGN4
in HFIP To confirm the effects of HFIP on the structure of GGN4, we measured steady-state fluorescence emis-sion spectra of fluorescein-labeled GGN4 (GGN4-F*). At higher HFIP concentrations, the emission intensity of GGN4-F* increased with a concomitant blue-shift in emission maxima (Fig. 5). The inset of Fig. 5 gives the emission maxima of GGN4-F* and those of free dye (FITC) at different HFIP concentrations. While the blue-shift of free dye was minimal (Δλ= 3 nm) and linear, the blue-shift of GGN4-F* was substantial (Δλ= 9 nm) and sigmoidal, which points to a cooperative conforma-tional transition at higher HFIP concentrations. Estimation of GGN4-induced pore sizes in planar lipid
bilayers Recent electrochemical data suggest the presence of GGN4-induced cation-selective pores in planar lipid bilayers (Kim et al., 1999). To estimate the size of the GGN4 pores in artificial lipid bilayers, we used organic cations of different sizes. Figure 6 illustrates the current-
A
B
Fig. 4. Equilibrium sedimentation analysis of GGN4 in 50%
EtOH. The distributions of the absorbance (lower graph) of
GGN4 at 220 nm (square) and 230 nm (circle) at ultracentrifu-
gal equilibrium at 30,000 revs/min 25
°C in 2.5 mM potassium
phosphate buffer, pH 3.3 containing 50% EtOH. The distribu-
tions of the residuals (upper graph) as functions of the radial
position are also shown. The fitted lines are for a thermody-
namically ideal monomer fitted simultaneously to the data at the
two different wavelengths (220 and 230 nm).
voltage relation recorded under near-biionic conditions: 200 mM tetraethyl ammonium chloride (TEA-Cl) or N-methyl-D-glucamine (NMDG) and 200 mM KCl in the recording pipette. The respective permeability ratios of TEA+ and NMDG+ to K+ ions (PTEA/PK and PNMDG/PK), calculated from the GHK equation using the mean rever-sal potentials shown in Fig. 6, were 0.42 and 0.041. Al-though the channels formed by GGN4 displayed lower permeability to TEA+ or NMDG+ than to K+, the genera-tion of inward current by TEA+ or NMDG+ strongly sug-gests that the GGN4-induced pores were large enough for these large organic cations. Therefore, our results indicate that the diameter of the GGN4-induced pores must be at least 7.3 Å, since the geometrical mean diameters of TEA+ and NMDG+ are 6.6 and 7.3 Å, respectively (Villar-roel et al., 1995).
Discussion
Although they differ widely in length and sequences, the amphipathic α-helices in various antibiotic peptides are important. The CD analyses in this and previous work (Park et al., 2000) demonstrated that GGN4 forms helical structures in membrane-mimicking environments. MeOH,
234 Oligomeric State of Gaegurin 4
Fig. 5. Fluorescence spectra of fluorescein-labeled GGN4 at
increasing concentrations of HFIP. Numbers indicate concentra-
tions of HFIP. Excitation wavelength was 490 nm. The concen-
tration of peptide was ~2 × 10-6 M. Inset: effects of HFIP con-
centration on the emission maximum of fluorescein-labeled
GGN4 (open square), and free fluorescein dye (FITC) (open
circle). The dye emission maxima are fitted to a linear function
and those of GGN4-F* to a sigmoidal function.
EtOH and HFIP each increased the helical content of GGN4, but to different degrees, in the order: 15% HFIP > 50% EtOH > 50% MeOH (Fig. 1A). Based on the CD data (Fig. 1B), GGN4 formed a helix from approximately 6% HFIP, which is close to the fluorescence emission transition point (Fig. 5). In addition, the fluorescence emission maxima of GGN4-F* blue-shifted in a sigmoidal manner, which points to a cooperative transition. An HFIP-induced cooperative transition of spin-labeled ce-cropin to a higher-order oligomer has also been reported. (Mchaourab et al., 1993).
Some antimicrobial peptides with the potential to form amphipathic α-helical structures aggregate into higher oligomeric structures in a lipid bilayer (He et al., 1996; Matsuzaki, 1995). In this study, we demonstrated that GGN4 underwent a coil to helix transition and formed oligomers of a maximum size correponding to decamers (10-mers) in 15% HFIP (Fig. 3). Because HFIP is one of the most potent helix enhancers, decamers may be the largest aggregates that GGN4 can form. However, if HFIP forms micells and the co-solvent binds to GGN4, the GGN4 aggregates may be smaller than this estimate. In order to understand the exact influence of HFIP on GGN4 aggregation, further equilibrium sedimentation analyses, which view the HFIP-GGN4 interactions as peptide-detergent complex systems (Fleming et al., 1997), are underway. Similar sizes of pore were observed with alamethicin, which forms 8 to 11-mer barrel-stave-type aggregates, depending on conditions (He et al., 1996), and magainin 2, which forms supramolecular structures of 4 to 7-mer peptide/lipid toroidal-type pores (Matsuzaki et
al., 1995; 1996). The relationship between peptide aggre-gation state and antibiotic activity is not yet understood.
Fig. 6. GGN4-induced current features. GGN4 currents were
evoked by ramp commands at 200 mM bath N-methyl D-
glucamine [NMDG, CH3NHCH2(CHOH)4CH2OH, Mw = 195.22]
and tetraethylammonium chloride (TEA-Cl). Reversal potentials
at 200 mM NMDG+ and TEA+ against K+ in the patch pipette
were -41.31 ± 2.86 (N = 3), and -17.96 ± 1.44 mV (N = 5), re-
spectively. The permeability ratios for NMDG+ and TEA+
against K+ were 0 and 0.374, respectively.
However, recent studies indicate that oligomerization of peptides can have dramatic effects on antibacterial action (Feder et al., 2000) and membrane leakage properties (Mazzuca et al., 2005).
An estimate of the GGN4 pore size in lipids was also obtained from the electrochemical data (Fig. 6). From the patch-clamp analysis, the internal diameter of the pores in the lipid bilayer was estimated to be larger than 7.3 Å in order for the NMDG+ ion to pass through the channel. From this we were able, with the following equation (Ma-tsuzaki et al., 1996), to estimate the lower limit of the oligomeric state of the GGN4 pore: D = 2*r*[(1/sin(π/np)) -1] (Rinaldi, 2002) where, D is the internal diameter of the pore, r the radius of the peptide helix which was approximated at 5 Å, and np the number of peptide molecules. From this equation we estimated the internal diameters of the tetrameric (np = 4) and pentameric (np = 5) pores to be 5.4 Å and 8.7 Å, respectively. Therefore, in order to allow the NMDG+ ion (7.3 Å) to pass through the channel, the pore has to be generated by at least a pentamer (5-mer), assuming a bar-rel-stave type GGN4 pore. However, considering that lipid membranes, as well as peptide-lipid interactions, are dynamic (Matsuzaki et al., 1996), the appearance of GGN4 pores may be transient and too unstable to com-prise a unique form of homogeneous oligomer. This pos-sibility is also reflected in a recent study (Kim et al., 1999a; 2004) of the voltage-dependence and cation selec-tivity of GGN4 channels, which showed that these chan-nels are highly heterogeneous in their conductance and
Su-Yong Eun et al. 235
gating. It is likely that the GGN4 pores are also heteroge-
neous in size and transient in their nature in a real mem-
brane environment due to the fluidity of the lipid bilayer
and the complex cellular environment.
In summary, we have demonstrated that GGN4 is a
helical peptide that can form oligomeric structures in lipid
membranes as well as in membrane-mimicking environ-
ments. We estimated the minimal and maximal sizes of
the pores as pentamers (5-mers) and decamers (10-mer),
using analytical ultracentrifugation and patch-clamp tech-
niques, respectively. The CD and fluorescence data dem-
onstrated that the oligomeric transition was cooperative
and accompanied by a large secondary structure change
from a near random coil to a helix.
Acknowledgments This study was supported by a grant from
the Biomedical Brain Research Center of the Korea, Health 21
R&D Project, funded by the Ministry of Health & Welfare, Re-
public of Korea (A040042), to S.-Y. Eun, and an intramural
grant from Mokpo National University to S.-J. Kim.
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