NMR spectroscopic assessment of the structure and dynamic properties of an amphibian antimicrobial peptide (Gaegurin 4) bound to SDS micelles.

Abstract

The structure and dynamics of a 37-residue antimicrobial peptide gaegurin 4 (GGN4) isolated from the skin of the native Korean frog, Rana rugosa, was determined in SDS micelles by NMR spectroscopy. The solution structure of the peptide in SDS micelles was determined from 352 NOE-derived distance constraints and 22 backbone torsion angle constraints. Dynamic properties for the amide backbone were characterized by (1)H-(15)N heteronuclear NOE experiments. The structural study revealed two amphipathic helices spanning residues 2-10 and 16-32 and that the helices were connected by a flexible loop. An intraresidue disulfide bridge was formed between residues Cys31 and Cys37 near the C-terminus. The loop region (11-15) connecting the two helices are were slightly more flexible than these helices themselves. From the fact that since there is no contact NOEs between two helices, it is implied that the GGN4 peptide shows an independent motion of both helices which has an angle of about 60 degrees -120 degrees from each other.

Full text

Page 1

Journal of Biochemistry and Molecular Biology, Vol. 40, No. 2, March 2007, pp. 261-269
NMR Spectroscopic Assessment of the Structure and Dynamic Properties
of an Amphibian Antimicrobial Peptide (Gaegurin 4) Bound to SDS Micelles
SangHo Park1,2, Woo-Sung Son2, Yong-Jin Kim2, Ae-Ran Kwon2 and Bong-Jin Lee2,*
1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA
2Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University,
San 56-1, Shillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea
Received 18 September 2006, Accepted 29 November 2006
The structure and dynamics of a 37-residue antimicrobial
peptide gaegurin 4 (GGN4) isolated from the skin of the
native Korean frog, Rana rugosa, was determined in SDS
micelles by NMR spectroscopy. The solution structure of
the peptide in SDS micelles was determined from 352
NOE-derived distance constraints and 22 backbone
torsion angle constraints. Dynamic properties for the amide
backbone were characterized by 1H-15N heteronuclear
NOE experiments. The structural study revealed two
amphipathic helices spanning residues 2-10 and 16-32 and
that the helices were connected by a flexible loop. An intra-
residue disulfide bridge was formed between residues
Cys31 and Cys37 near the C-terminus. The loop region
(11-15) connecting the two helices are were slightly more
flexible than these helices themselves. From the fact that
since there is no contact NOEs between two helices, it is
implied that the GGN4 peptide shows an independent
motion of both helices which has an angle of about 60o-120o
from each other.
Keywords: Antimicrobial peptide, Dynamics, Gaegurin,
Micelle, NMR, Structure
Introduction
Antimicrobial peptides have been attributed pivotal roles in
innate immune responses of living organisms (Gabay, 1994).
Many different families of antimicrobial peptides, as based on
their amino acid sequence and secondary structure, have been
isolated from insects, plants, animals, and microorganisms
(Boman, 1995; Nicolas and Mor, 1995). In particular, several
peptides from the skin of the amphibian have been found to
possess broad-spectrum antimicrobial activities (Zasloff,
1987; Gibson et al., 1991; Mor et al., 1991; Simmaco et al.,
1991; Morikawa et al., 1992; Simmaco et al., 1993; Clark et
al., 1994). Thus, antimicrobial peptides are a promising source
of new antibiotics for combating the increasing emergence of
drug-resistant bacteria.
Gaegurins (GGNs) have been isolated from the skin of the
native Korean frog Rana rugosa. These peptides show a broad
spectrum of antimicrobial activity against various microorganisms
but they appear to have little to no hemolytic properties
(Simmaco et al., 1991). The GGNs are divided into two
families based upon their amino acid sequence. Thus, Family
I includes GGN1 to GGN4, which are composed of 33 to 37
amino acids whereas Family II includes GGN5 and GGN6,
which consist of 24 amino acids and contain proline at
position 14 near the center of the sequence. All six GGN
peptides contain two invariant cysteine residues, one at the C-
terminus and the other at position 7 from the C-terminus. The
heptapeptide motif containing these two cysteine residues is
linked by an intra-residue disulfide bridge, which has been
conserved in the antimicrobial peptides derived from other
genus Rana such as the brevinins, esculentins and ranalexin
(Zasloff, 1987; Morikawa et al., 1992; Simmaco et al., 1993).
Of the six GGN peptides, GGN4 is most abundant in the frog
skin and is believed to play the most important role in the
innate defense system of the frog (Park et al., 1994).
Antimicrobial peptides, which adopt one or two amphipathic
helices and act directly on the membrane of their target cells,
have been studied in different chemical environments: organic
solvent-water mixtures, detergent micelles, and lipid bilayers
(Bechinger et al., 1993; Gesell et al., 1997; Vignal et al.,
1998; Park et al., 2000; Park et al., 2002; Won et al., 2002). It
has been reported that the structure of several membrane
binding peptides determined in organic solvents are similar to
those that have been determined in detergent micelles (Gesell
et al., 1997; Vignal et al., 1998; Won et al., 2002). Although
*To whom correspondence should be addressed.
Tel: 82-2-880-7869; Fax: 82-2-872-3632
E-mail: lbj@nmr.snu.ac.kr

Page 2

262SangHo Park et al.
organic solvents mimic membrane environments and generally
induce the structure of membrane binding proteins and
peptides, detergent micelles are regarded as a more adequate
environment for membrane binding proteins. Since actual
membranes consist of lipid bilayers to form a heterogeneous
environment in which their outer head group is polar and their
inner acyl group is apolar, detergent micelles can also provide
heterogeneous environments characterized by polar head
groups and apolar acyl tails. The relatively recent development
and application of multidimensional NMR spectroscopy for
studies of membrane peptides and proteins takes advantage of
the well characterized model membrane environments of
detergent micelles (Gesell et al., 1997; Vignal et al., 1998;
Park et al., 2002; Won et al., 2002; Leetachewa et al., 2006).
Detergent micelles that have been widely used in solution
NMR studies of membrane proteins include sodium dodecyl
sulfate (SDS), dodecylphosphocholine (DPC), and dihexanoyl-
phosphatidylcholine (DHPC).
We have determined the structure of the GGN4 peptide
bound to SDS micelles using heteronuclear NMR spectroscopy
and compared that to GGN4 binding in a 50% trifluoroethanol
(TFE) solution which had been reported previously (Park et
al., 2000). Since GGN4 in 50% TFE solution revealed an
undefined flexible loop region connecting the two helices, we
investigated the backbone dynamics of GGN4 when bound to
SDS micelles by using steady-state 1H-15N NOE values. We
propose that these results may provide more precise
information on the elucidation and understanding of structure-
activity relationships of the GGN4 peptide.
Materials and Methods
Materials. 15NH4Cl was obtained from Isotec (Miamisburg) while
SDS-d25 and DL-1, 4-dithiothreitol-d10 were purchased from Cambridge
Isotope. 99.95% D2O, thrombin, carboxypeptidase Y, and
phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma.
Sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) was obtained
from Aldrich. All other chemicals were analytical grade and were
obtained from several different manufacturers.
Sample preparation. The E. coli strain DH5α containing the
plasmid encoding GST-fused GGN4 was grown in tryptone broth
with glucose (TBG) to yield non-labeled GGN4. Uniformly 15N
labeled GGN4 was obtained by growing the bacteria in M9
minimal media with 15NH4Cl as the sole nitrogen source. For 15N-
Leu and 15N-Lys selectively labeled GGN4 samples, the medium
was supplemented with 200 mg/l of each of the 19 amino acids
except for 100 mg/l of the desired 15N-labeled amino acid. The
purification method used for GGN4 was slightly modified from that
cited previously (Park et al., 1994; Park et al., 2000). In brief,
suspended in 50 ml of 0.5 M NaCl, 20 mM Tris-HCl (pH 8.0), per
liter of culture and disrupted by sonication (duty cycle 30%, output
control 5, Branson Sonifier 450) for 10 min on ice, and then
centrifuged at 20,000 × g for 30 min at 4oC. The supernatant was
discarded and the pellet resuspended in a binding buffer (0.5 M
NaCl, 5 mM imidazole, 20 mM Tris-HCl, 0.5% sarkosyl, pH 8.0).
The fusion protein was cleaved by exposure to a final concentration
of 10 µM thrombin for a period of 12 h at 30oC. The resulting His-
tagged GGN4 was purified using nickel affinity chromatography
(His · Bind Resin) followed by lyophilization. The resulting white
powder (His-tagged GGN4) was dissolved in 70% formic acid to a
final concentration of 10 mg/ml, and then a 10-fold molar excess of
cyanogen bromide (CNBr) was added to the solution. The reaction
was kept in the dark for 24 h at room temperature and the reaction
mixture then evaporated to dryness and subsequently dissolved in a
95% water/5% acetonitrile mixture. The recombinant GGN4 was
purified by semi-preparative reverse-phase HPLC on a Lichrospher
C18 column (10 µ, 100Å, 4 × 250 nm, Merck) using a water-
acetonitrile gradient. The purity and the primary structure of GGN4
were confirmed by SDS-PAGE (Fig. 1) and matrix-assisted laser
desorption/ionization (MALDI) mass spectroscopy. GGN4 prepared
by this method yielded a peptide with the same bioactivity as
GGN4 isolated directly from frog skin.
NMR spectroscopy. All NMR experiments were performed on
Bruker DRX 500 and 600 MHz spectrometers equipped with a
gradient unit (Park et al., 2003). Samples for NMR measurements
were prepared from lyophilized preparations so as to make a 2.0
mM solution in 500 mM SDS-d25 (90% H2O, 10% D2O), pH 3.0.
The 1H-15N HSQC spectra were monitored over an increasing
temperature range (from 30oC to 60oC by 5 degree increments).
Maximal spectral resolution was obtained at 50oC; therefore,
subsequent NMR experiments were performed at 50oC (Fig. 2). A
3-dimensional (3D)
recorded with an isotropic mixing time of 60 ms and 3D 15N-edited
NOESY-HSQC spectra with 150 and 250 ms mixing times. Water
resonance was suppressed by using a WATERGATE sequence.
Slowly exchanging amide protons were monitored with a series of
1H-15N HSQC spectra. The 3JHNHα coupling constants were measured
15N-edited TOCSY-HSQC spectrum was
Fig. 1. Protein fractions at different stages of the purification
protocol of GGN4 visualized by 15% tricine gel electrophoresis.
Lane 1: total cell before IPTG induction, Lane 2: total cell after
IPTG induction with the fusion peptide band at 31 kDa. Lane 3:
mixture after thrombin digestion, Lane 4: His-tagged GGN4
purified by Ni-affinity chromatography, Lane 5: mixture after
CNBr cleavage, Lane 6: purified GGN4 by RP-HPLC. The
arrow indicates the band of GST, His-tagged GGN4, and GGN4
from top to bottom.

Page 3

Structure and Dynamics of GGN4263
from the 2D HMQC-J experiment (Kay and Bax, 1990). The 1H-
15N steady-state NOE values were determined from spectra
recorded in the presence and absence of a proton presaturation
period of 3 s (Farrow et al., 1994). 1H saturation was achieved with
the use of 120o 1H pulses applied every 5 ms. In the case of the no
NOE spectra (equilibrium nitrogen magnetization spectra), a net
relaxation delay of 5 s was employed, while a relaxation delay of
2 s prior to a 3 s proton presaturation period was employed for the
NOE spectra. The 2D TOCSY, NOESY, and DQF-COSY spectra
were also acquired for a non-labeled sample. The 2D TOCSY
spectra were acquired using an MLEV-17 spin lock sequence with
isotropic mixing times of 30 and 60 ms, respectively. The NOESY
spectra were acquired with mixing times of 150, 200, and 250 ms,
respectively. For the DQF-COSY experiments, solvent suppression
was achieved using selective low-power irradiation of the water
resonance. Solvent suppression for the NOESY and TOCSY
experiments was achieved by using the WATERGATE sequence.
The spectra were processed on a Silicon Graphics Indigo-2
workstation, using the NMRPipe program (Delaglio et al., 1995).
Chemical shifts were referenced to the methyl signal of DSS.
Structure calculations. Distance restraints were obtained from the
homonuclear and heteronuclear NOESY spectra with 200 ms
mixing times. Comparisons were made to the 150 and 250 ms
NOESY spectra to assess possible contributions from spin
diffusion. All NOE data were classified into three classes: strong,
medium, and weak, corresponding to upper bound interproton
distance restraints of 3.0, 4.0, and 5.0 Å, respectively. Lower
distance bounds were taken as the sum of the van der Waals radii of
1.8 Å. As no stereospecific assignment could be made for the
methyl and methylene protons, appropriate pseudo atom corrections
were applied (Wüthrich et al., 1983). A total of 352 NOE
constraints, 22 backbone dihedral angle restraints, and 28 hydrogen
bond restraints were included in the calculation of the structures. In
addition, three other restraints were added to define the disulfide
bridge between Cys31 and Cys37. Thus, the target values of S(31)-
S(37), S(31)-C(37), and S(37)-C(31) were set to 2.20 (± 0.02) Å,
2.99 (±0.5) Å, and 2.99 (±0.5) Å, respectively (Nilges et al., 1988).
The structure calculations of the peptides were performed using
repeated iterative cycles of ab initio simulated annealing using the
force field adapted for NMR structure determination (parallhdg.pro)
in XPLOR 3.851 (Brünger, 1992). Simulated annealing was
performed from an extended conformation for 30 ps (time step = 5
fs) with an initial annealing temperature of 1,000 K, which was
followed by 15 ps (time step = 5 fs) cooling steps to 100 K.
Refinement of the structures was performed using the conjugate
gradient Powell algorithm with an initial annealing temperature of
300 K and 5,000 cycles of energy minimization, using the same
force field file (parallhdg.pro) with ab initio simulated annealing
protocol. A total of 48 structures of the peptides, with no distance
violation larger than 0.5 Å and no dihedral violation larger than 5o,
were accepted from the 50 structures that were generated. The 20
best structures of the peptides determined on the basis of their total
energy and with no systematic distance violation greater than 0.3 Å
and no dihedral angle violation larger than 3o were selected as the
final structures of the peptides.
Results
Purification of GGN4. The overexpression of a protein is a
prerequisite for efficient heteronuclear NMR studies, however,
it is difficult to over express GGN4 directly in E. coli due to
the innate toxicity towards the microbe. To obtain the
relatively large quantities of peptide needed and produce the
peptide without killing the E. coli, GGN4 was constructed and
expressed as a fusion peptide utilizing the glutathione-S-
transferase (GST)-fusion system (Park et al., 1994). The GST-
GGN4 fusion protein was successfully over expressed in E.
coli (Fig. 1, lane 1 and 2) and subsequently purified efficiently
as inclusion bodies. After thrombin directed cleavage of the
fusion protein, His-tagged GGN4 was purified by Ni affinity
chromatography and then cleaved with CNBr to release intact
GGN4 peptide (Fig. 1, lanes 3-6). Typically, 2 mg of GGN4
could be obtained from 1L of cell culture in uniformly 15N
labeled minimal media. The homogeneity of the purified
peptide was confirmed by MALDI TOF mass spectroscopy.
NMR spectroscopy of GGN4 in SDS micelles. On the basis
of the assignment results of GGN4 previously obtained in a
50% TFE solution (Park et al., 2000), the assignment of
backbone atoms of GGN4 in SDS micelles was mainly
performed by conducting the heteronuclear experiments in
combination with the method proposed by Wüthirch (Wüthrich,
1986). Since the molecular weight of GGN4 bound to SDS
micelles increased to about 25 kDa, the overall NMR signals
were broadened when compared to the signals in a 50% TFE
solution so that several peaks overlapped. To optimize the
spectral resolution, we monitored the 1H-15N HSQC spectra of
GGN4 in SDS micelles by 5 degree step-wise increments of
temperature (from 30oC to 60oC) (Fig. 2). We achieved
maximal spectral resolution at 50oC whereby the appropriate
number of backbone and side chain resonances were present
Fig. 2.
micelles monitored at different temperatures. (A) 35oC, (B) 40oC,
(C) 45oC, (D) 50oC. Several peaks resolved at 50oC are indicated
by the arrows.
1H-15N HSQC spectra of GGN4 in 500 mM SDS

Page 4

264SangHo Park et al.
without evidence of resonance doubling indicative of multiple
species contributing to conformational heterogeneity (Fig.
2D). Although there is limited dispersion in the 1H chemical
shift dimension typical of a helical peptide, all of the backbone
resonances were resolved and assigned as indicated.
The sequential connections were compared with dαN (i,
i + 3), dαN (i, i+4), and dαβ(i, i+3) connections to ensure their
assignments. Assignment ambiguities due to peak overlaps
were resolved by selectively using 15N Leu and Lys labeled
samples of the 1H-15N HSQC spectra. Figure 3 demonstrates
1H(ω1), NH(ω3) cross sections obtained from a 3D 1H-15N
NOESY-HSQC spectrum of GGN4 extracted at different 15N
amide frequencies (ω2). The sequential connection was
represented from residues Phe9 to Asp16, which contained the
flexible loop region (Lys11-Lys15). The chemical shift values
of proton and 15N resonances of GGN4 in a 500 mM SDS
micelle are presented in Table 1.
The short and medium inter-residue NOE cross-peaks
identified by this study and their relative intensities are
presented in Fig. 4. The NOE connectivity’s of GGN4 in SDS
micelles are similar with those cited for the 50% TFE
solution. This observation indicates nearly complete sets of
dNN (i, i + 1), dαβ(i, i + 3), dαN (i, i + 4), and dαN (i, i + 3) NOE
connectivities for residues 2 to 10 and 16 to 32, and suggests
the presence of a regular α-helical conformation.
coupling constants for residues 2-10 and 16-32 show almost
less than 5.5 Hz except for the residues, Gln8, Phe9, Gly20,
Gly24, and Cys31. Further support for helical regions to exist
between residues 2-10 and 16-32 was provided by the amide
proton exchange data. Slowly exchanging amide protons were
observed in the central residues of the helical segments
spanning residues 6-10 and 20-32. The chemical shifts of the
α-protons of residues 2-10 and 16-32 showed an overall up
field shift tendency by 0.1 to 0.4 ppm compared to random
coil chemical shifts, which also supports the notion of the
helical conformation of GGN4 (Wishart et al., 1992). The
intra-residue disulfide bond between Cys31 and Cys37 can be
inferred from the presence of the NOE cross-peaks of
Cys31Hβ-Cys37Hβ, Cys31NH-Cys37Hβ, and Cys31Hβ-Cys37NH.
3JHNHα
Three-dimensional structure and dynamic properties. A
set of 50 structures of GGN4 was calculated by using 352
distance restraints, 22 dihedral angle restraints, and 28 hydrogen
bond restraints. Simulated annealing (SA) calculations were
performed to produce structures with a common fold that
were in acceptable agreement with the experimental restraints
and with low total energies. Of the 50 structures, 48 with no
distance violation greater than 0.5 and no dihedral violation
larger than 5o were selected. The 20 structures with the lowest
energies were then chosen to represent the solution structure
of the GGN4 peptide. Among these 20 structures, three were
considered as representative and were depicted by two locally
well-defined helices comprised of residues 2 to 10 on one
hand and residues 16 to 32 on the other (Fig. 5). The 20
structures chosen revealed that there is no contact between the
helices thereby allowing an independent movement of both
helices at an angle of 60o-120o to each other.
The dynamic behavior of the GGN4 peptide bound to SDS
micelles was probed by the heteronuclear
experiment. The experimental 1H-15N heteronuclear NOEs for
all backbone amide sites of the membrane-bound GGN4 are
shown in Fig. 6. The regions with the longest correlation
times, as reflected in the positive 1H-15N heteronuclear NOE
values, generally correlated with the locations of the helices.
The N- and C-terminal residues are substantially more flexible
than are the residues within the helices. The loop region (11-
15) connecting the two helices are slightly more flexible than
the helices.
Structural statistics for the mean and 20 converged structures
were evaluated in terms of structural parameters (Table 2).
The 20 final converged structures were shown to exhibit an
RMSD about the mean coordinate positions for residues 2-10
and for residues 16-32 and were 0.21 and 0.61 for their
backbone atoms, respectively.
1H-15N NOE
Fig. 3. Series of strips selected from a 3D
HSQC spectrum of GGN4 in the 500 mM SDS micelles. The
spectrum was recorded at 50oC with a mixing time of 200 ms
and extracted at the 15N chemical shifts of residues Phe9 to
Asp16. Intra-residual contacts are labeled with symbols.
Sequential NOE contacts are represented by arrows.
1H-15N NOESY-

Page 5

Structure and Dynamics of GGN4265
Discussion
As a result of this study, we have been able to determine the
structure of the GGN4 peptide in SDS micelles. Insofar
detergent micelles are regarded as appropriate materials than
organic solvents for studying membrane peptides and proteins
by solution NMR spectroscopy (Nelson and Kallenbach,
1986; Park et al., 2002; Roccatano et al., 2002; Maeda et al.,
2003).
The structure of GGN4 in SDS micelles was shown to be
similar to that using 50% TFE solvents. Our results are also in
agreement with the CD studies of GGN4 showing that the
peptide forms mainly an α-helix in 50% TFE/water solvents,
DPC micelles, and SDS micelles, and that the helix content
estimated under these conditions almost same to be about
70% (Park et al., 2000). Alternatively, GGN5 another
antimicrobial peptide (of the Family II of GGN) and several
of its derivatives, have a 5-10% higher helix content in 50%
TFE/water solvents than in SDS and DPC micelles which
suggests that TFE may induce or stabilize helical structures
(Park et al., 2002). However, determination of their structures
by NMR spectroscopy in SDS micelles showed a more helical
Table 1. 1H and 15N resonance assignment of GGN4 in 500 mM SDS micelles at 50oC, pH 3.0
Residue
15NHNαHβHγHothers
Gly1
Ile2
Leu3
Asp4
Thr5
Leu6
Lys7
Gln8
Phe9
Ala10
Lys11
Gly12
Val13
Gly14
Lys15a
Asp16
Leu17
Val18
Lys19a
Gly20
Ala21
Ala22
Gln23
Gly24
Val25
Leu26
Ser27
Thr28
Val29
Ser30
Cys31
Lys32
Leu33
Ala34
Lys35
Thr36
Cys37
125.6
124.1
120.3
122.1
125.3
122.1
120.8
124.2
125.1
119.9
111.0
125.0
111.8
123.6
121.0
125.0
121.0
123.6
110.6
128.3
123.6
120.0
112.6
125.9
123.7
118.3
122.6
124.2
117.0
122.2
127.8
123.1
122.5
117.3
112.4
126.7
8.71
8.17
7.92
7.92
8.33
8.30
7.80
8.22
8.51
8.02
7.93
8.00
8.47
8.11
7.96
8.26
7.95
8.11
8.16
8.19
8.34
8.10
8.22
8.11
8.32
8.21
7.83
8.28
8.26
8.03
8.29
8.05
7.82
7.85
8.40
7.82
4.09
4.12
4.49
4.05
4.18
3.94
4.11
4.55
3.94
3.98
3.98
3.91
3.71
4.01
4.52
4.25
3.88
4.01
4.00
4.35
4.13
4.10
4.07
3.93
4.10
4.29
4.40
3.72
4.15
4.68
4.25
4.25
4.40
4.22
4.53
4.71
1.99
1.86, 1.86
3.03, 3.03
4.35
1.97, 1.97
2.03, 2.03
2.24, 2.24
3.30, 3,30
1.55
1.97, 1.97
1.66, 1.34
1.68
γCH3/δCH3 1.02
δCH3 1.01, 1.01
1.27
1.70
1.72, 1.72
NOb
δCH3 0.95, 0.95
δCH2 1,45, 1.45
δNH2 7.38, 6.73
2,6H/3,5H 7.25, 7.24
1.82, 1.82δCH2 1.70, 1.48
2.110.89, 0.89
1.96, 1.96
3.16, 2.98
1.95, 1.95
2.26
1.96, 1.96
1.63, 1.63δCH2 1.49, 1.49
1.64
1.09, 0.93
1.63, 1.63
δCH3 1.12, 0.94
δCH2 1.49, 1.49
1.54
1.54
2.24, 2.242.52, 2.52δNH2 7.28, 6.80
2.27
1.87, 1.74
4.08, 4.08
4.40
2.26
4.03, 4.03
3.49, 3.14
2.15, 1.89
1.91, 1.91
1.57
2.24, 2.06
4.22
3.72, 3.03
1.14, 1.00
NOδCH3 1.11, 0.99
1.29
0.99, 0.99
1.69, 1.55
1.63
NO
δCH3 0.96, 0.96
1.80, 1.52
1.19
NO
aOverlap between K15 and K19; bNO, not observed