Designer nanorings with functional cavities from self-assembling β-Sheet peptides

Article abstract of DOI:10.1002/asia.201000428

β-Barrel proteins that take the shape of a ring are common in many types of water-soluble enzymes and water-insoluble transmembrane pore-forming proteins. Since β-barrel proteins perform diverse functions in the cell, it would be a great step towards developing artificial proteins if we can control the polarity of artificial β-barrel proteins at will. Here, we describe a rational approach to construct β-barrel protein mimics from the self-assembly of peptide-based building blocks. With this approach, the direction of the self-assembly process toward the formation of water-soluble β-barrel nanorings or water-insoluble transmembrane β-barrel pores could be controlled by the simple but versatile molecular manipulation of supramolecular building blocks. This study not only delineates the basic driving force that underlies the folding of β-barrel proteins, but also lays the foundation for the facile fabrication of β-barrel protein mimics, which can be developed as nanoreactors, ion- and small-molecule-selective pores, and novel antibiotics. Copyright

Full text of DOI:10.1002/asia.201000428

FULL PAPERS
DOI: 10.1002/asia.201000428
Designer Nanorings with Functional Cavities from Self-Assembling b-Sheet
Peptides
Il-Soo Park,[a] You-Rim Yoon,[a] Minseon Jung,[b] Kimoon Kim,[b] SeongByeong Park,[c]
Seokmin Shin,[c] Yong-beom Lim,*[d] and Myongsoo Lee*[a]
Dedicated to Professor Eiichi Nakamura on the occasion of his 60th birthday
Abstract: b-Barrel proteins that take
the shape of a ring are common in
many types of water-soluble enzymes
and water-insoluble transmembrane
pore-forming proteins. Since b-barrel
proteins perform diverse functions in
the cell, it would be a great step to-
wards developing artificial proteins if
we can control the polarity of artificial
b-barrel proteins at will. Here, we de-
scribe a rational approach to construct
b-barrel protein mimics from the self-
assembly of peptide-based building
blocks. With this approach, the direc-
tion of the self-assembly process
toward the formation of water-soluble
b-barrel nanorings or water-insoluble
transmembrane b-barrel pores could be
controlled by the simple but versatile
molecular manipulation of supramolec-
ular building blocks. This study not
only delineates the basic driving force
that underlies the folding of b-barrel
proteins, but also lays the foundation
for the facile fabrication of b-barrel
protein mimics, which can be devel-
oped as nanoreactors, ion- and small-
molecule-selective pores, and novel an-
tibiotics.
Keywords: nanostructures
·
pep-
tides · protein folding · proteins ·
self-assembly
Introduction
structures,[1–4] has been intense and is expected to escalate
further.[2,3,5–7] Artificial bionanostructures can mimic or even
have enhanced functional properties over the bionanostruc-
tures of biological origin (e.g., proteins and many subcellu-
lar organelles). Moreover, we can expect that artificial bio-
In recent years, an interest in manmade or artificial biona-
nostructures, including peptide-based self-assembled nano-
AHCTUNGTREGnNNUN anostructures would have properties that are unprecedent-
[a] I.-S. Park, Y.-R. Yoon, Prof. M. Lee
Center for Supramolecular Nano-Assembly
and Department of Chemistry, Seoul National University
Seoul 151-747 (Korea)
ed in nature. Since the major driving force that underlies
the formation of bionanostructures is a noncovalent self-as-
sembly process, elaborately designed synthetic self-assembly
building blocks should be one of the most suitable candi-
dates for the construction of artificial bionanostructures.
b-Barrel proteins are commonly found in many cytoplas-
mic and transmembrane proteins. Cytoplasmic b-barrel pro-
teins are water-soluble, whereas transmembrane b-barrel
proteins are water-insoluble amphiphiles. Two of the most
well-known examples of water-soluble b barrels are triose-
phosphate isomerase (TIM) barrel folds and b-barrel folds
of a green fluorescent protein family.[8] Transmembrane b-
barrel proteins are typically found in porins and other pro-
teins that span cell membranes.[9] There is a variety of func-
tions that transmembrane b-barrel proteins can perform,
and the functional categories are still expanding. Structural-
ly, a b barrel is a closed structure in which the first and the
last b strands in a polypeptide chain are hydrogen-bonded
Fax : (+82)2-889-1568
E-mail: myongslee@snu.ac.kr
[b] M. Jung, Prof. K. Kim
Center for Smart Supramolecules and Department of Chemistry
Pohang University of Science and Technology
Pohang 790-784 (Korea)
[c] S. Park, Prof. S. Shin
Department of Chemistry, Seoul National University
Seoul 151-747 (Korea)
[d] Prof. Y.-b. Lim
Translational Research Center for Protein Function Control
and Department of Materials Science and Engineering
Yonsei University, Seoul 120-749 (Korea)
Fax : (+82)2-312-5375
E-mail: yblim@yonsei.ac.kr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000428.
452
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Chem. Asian J. 2011, 6, 452 – 458

in an end-to-end manner. b strands in b-barrel proteins are
typically arranged in an antiparallel fashion.
hydrophilic oligoether dendron was attached to the side-
chain phenol group. As the tyrosine derivative was prepared
as an N-a-Fmoc-protected (Fmoc=9-fluorenylmethoxycar-
bonyl) amino acid, it could be incorporated into the growing
peptide chain by using standard Fmoc SPPS protocols.
Inspired by the transmembrane b barrels of biological
origin, many synthetic b-barrel-like assemblies have been
developed. Noteworthy examples include p-oligophenyl rod-
based b-barrel systems,[10–12] b-barrel helical pores from am-
phiphilic dendritic peptides,[13–15] b-sheet nanotubes from
cyclic peptides,[16] and supramolecular barrels from amphi-
philic rigid–flexible macrocycles.[17] These synthetic mole-
cules have shown the possibilities of constructing artificial
membrane pores or channels; however, their chemical com-
ponents and self-assembled structures are largely different
from those of natural b-barrel folds. In natural b-barrel
folds, b strands are composed of natural amino acids and lie
roughly parallel to the lipid bilayer membrane normal and
span the entire thickness of the membrane.[9] Herein, we de-
scribe the development of b-barrel protein mimics of dual
functionality through a b-sheet-forming peptide self-assem-
bly. The most unique feature of this system is the formation
of highly uniform and discrete water-soluble b-barrel nano-
Self-Assembling Peptide Nanorings in Aqueous Solution
To investigate the effect of bulky dendron placement within
the b-sheet-forming peptide, we first synthesized and tested
peptide T3 (Figure 1). A notable structural feature of pep-
tide T3 is the presence of a second-generation triethylene
glycol dendrimer at the central part of the peptide building
block. As the dendron is much bulkier than the b strand
(see the side view of peptide T3 in Figure 1), we anticipated
that the bulky dendrons would create the curvature at the
interface between each building block owing to the steric re-
pulsion between the dendritic chains. This building block is
quite different from block conjugates of polyethylene glycol
(PEG) and peptide since the ethylene glycol units of pre-
cisely defined molecular structure are placed in the middle
of the peptide segment.[23,24]
Investigation by circular dichroism (CD) spectroscopy
showed that peptide T3 forms b-sheet structures in aqueous
solutions (see the Supporting Information). The self-assem-
bled morphology of peptide T3 was visualized by transmis-
sion electron microscopy (TEM). Remarkably, peptide T3
formed discrete nanoring structures (Figure 1b and the Sup-
porting Information). Every nanostructure has the shape of
a nanoring, thereby indicating that the formation of nano-
rings from peptide T3 is a highly efficient process. The nano-
ACHTUNGTRENNUNGring structures with a hydrophobic interior that is highly
similar to natural b-barrel proteins in structure and composi-
tion, which can in turn be transformed into transmembrane
b-barrel pores by the simple manipulation of their molecular
structure.
Results and Discussion
The b-sheet ribbons are organized in such as way that each
b strand runs perpendicular to the 1D ribbon axis, which is
called a cross-b structure.[18,19] b barrels are in fact ring-
shaped nanostructures. To probe the structural requirements
for b-sheet peptide nanoring formation, we set out to find a
fundamental design principle for constructing nanoring
structures from b-sheet peptides. Considering the fact that
nanorings are highly curved structures, we hypothesized that
the induction of curvature between the adjacent b strands
would force the 1D structure to bend. On the basis of this
hypothesis, we designed T-shape b-sheet peptide building
blocks, thus anticipating that bulky hydrophilic dendrons
placed at the central part of b-sheet-forming peptides might
ring diameter is highly uniform (ꢀ11 nm), thereby indicat-
ing that there is a preferred geometrical packing require-
ment for nanoring formation. The cross-section of nanorings
was about 4 nm, which corresponds well with the width of
fully extended peptide T3 (3.8 nm by the Corey–Pauling–
Koltun (CPK) model; see Figure 1d). Therefore, these re-
sults suggest that the nanorings are composed of a single
layer of building blocks, with the b strands oriented perpen-
dicular to the plane of the nanoring to be accommodated
into a highly bent structure. A recent theoretical study in
the gas phase, which has suggested that the rolling up of a
b-sheet can drive the formation of a b barrel,[25,26] further
supports this view. This finding, together with the uniform
nanoring size, suggests that nanorings have been formed by
end-to-end connection of b strands similarly to natural b-
barrel proteins.
induce curvature at the interface between
(Figure 1 and the Supporting Information).
b strands
The organic/peptide hybrid T-shape building blocks con-
sist of a b-sheet-forming peptide and a hydrophilic oligoeth-
er dendron. The dendron is symmetrically placed on the
side face of the peptide backbone. The artificially designed
b-sheet peptide has a repeating structure of hydrophobic
(tryptophan), positively charged (lysine), hydrophobic (tryp-
tophan), and negatively charged (glutamic acid) amino
acids, which has been found to promote the correct hydro-
gen-bonding arrangement for b-sheet formation.[2,20–22] The
building blocks were synthesized by a solid-phase peptide
synthesis (SPPS) method with the combination of both natu-
ral amino acids and a modified amino acid. The modified
amino acid was made from l-tyrosine, in which a bulky and
Consistent with these observations, structure modeling by
molecular dynamics (MD) revealed that an approximately
11 nm-sized nanoring is the most stable structure for the
peptide T3 aggregates (Figure 1e). The configurations for
T3 and the minimized structure of the 40-mer toroidal struc-
ture are shown in Figure 1e. For smaller toroidal structures
(i.e., 12-mer and 24-mer), it was found that their initial
structures became unstable and were broken within 100 ps
MD simulations at 300 K. For the 40-mer of T3; however,
the toroidal pore structure was found to be stable and main-
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M. Lee, Y.-b. Lim et al.
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tained its structure up to 1 ns of
MD simulations. The simulation
shows that the diameter for the
inner pore is 3.5–4.5 nm, where-
as the whole structure has the
diameter of about 11 nm and
thickness of about 4 nm, which
is consistent with the experi-
mental observations. More de-
tailed simulation results will be
published elsewhere.
To corroborate the notion
that nanorings have not been
formed during solvent evapora-
tion process or by staining re-
agent while preparing the TEM
samples on a grid, cryo-TEM
and dynamic light scattering
(DLS) experiments were per-
formed. A cryo-TEM image in
the Supporting Information
shows dark toroidal objects of
peptide T3 against the vitrified
solution background, thereby
confirming that toroids have
been formed in bulk solution.
DLS experiments further corro-
borated this result. The DLS
result shows the formation of
nano-objects with a hydrody-
namic radius (RH) centered at
7.5 nm (Figure 1c). The small
difference in the aggregate size
between DLS and negative-
stain TEM data is likely as a
result of the samples being hy-
drated under the solution con-
dition of DLS, whereas the
sample is in a dried state during
negative-stain TEM investiga-
tion. The inset in Figure 1c
shows the FTIR spectrum of
peptide T3. The presence of
amide I contours for the b-
sheet doublet at 1631 and
1691 cmÀ1 reveals that the b-
sheet structure is in an antipar-
allel conformation within the
nanorings.[27] All these data sup-
port the conclusion that peptide
T3 forms stable and water-solu-
ble b-barrel nanorings. Consid-
ering the fact that b strands are
in fact tilted with respect to the
barrel axis in natural b-barrel
proteins, b strands in the pep-
tide nanorings are also likely to
Figure 1. a) Structures of T-shape peptides. b) Negative-stain TEM image of peptide T3 in 20 mm KF. c) Hy-
drodynamic radius distribution of peptide T3 (20 mm) aggregates in 20 mm KF. Inset: FTIR spectrum of pep-
tide T3. d) Corey–Pauling–Koltun (CPK) model of peptide T3 and the model of self-assembled b barrels.
e) MD simulation of T3. Minimized structure of the 40-mer T3 b barrels (left). Top view (middle) and side
view (right) of the resulting structure after 1 ns MD simulation.
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Designer Nanorings with Functional Cavities
be tilted to form flat b sheets. More in-depth atomic-level
structural studies should be the subject of the further re-
search.
secondary structure and forms huge irregular aggregates
(see the Supporting Information). These results can be inter-
preted in terms of geometric constraints; the highly bulky
dendrons induce excessive curvature at the interface be-
tween the peptides, which is not tolerable for a proper b-
sheet hydrogen-bonding arrangement. Altogether, these
studies indicate that there exists a certain optimal range of
dendron bulkiness in which the balance between the curva-
ture induction and the b-sheet formation is delicately con-
trolled.
Structural Requirements for Nanoring Formation
To gain further insight into the structural requirement for
nanoring formation, we synthesized analogous T-shape pep-
tides (T1, T2, and T4) while varying the relative volume
fraction (f) of a dendron to a b-sheet peptide. As peptide T1
contains an unmodified tyrosine, it has the smallest volume
fraction (fꢀ0). CD study reveals that peptide T1 forms a b-
sheet structure (Figure 2a, inset); however, peptide T1 only
forms fibers that are several micrometers in length with no
indication of nanoring structure formation (Figure 2a). The
width of the fiber (4.8 nm) corresponds well to the height of
peptide T1 (4.7 nm by the CPK model), thereby suggesting
that the fibers are typical bilayered b ribbons.[2,21,28] There-
fore, this result supports the view that the bulkiness of the
dendron segment and resulting steric repulsion effect are re-
sponsible for nanoring formation.
T-Shape Peptides in a Lipid Bilayer Environment
We next asked the question of whether or not the water-
soluble nanorings (b barrels) can be converted into hydro-
phobic transmembrane b barrels by simple variation in mo-
lecular structure. As the thickness of the nanoring (ꢀ4 nm)
is large enough to span common lipid bilayer membranes
(2–4 nm),[31] the transmembrane b barrels, if successfully
formed, might act as pores or channels. Studies have shown
that, when appropriately designed, various types of organic
and inorganic molecules can perform the function of trans-
membrane pores and channels.[11,15,16,32,33] To enable the
outer surface of b barrels to interact with the inner hydro-
phobic space of a lipid bilayer membrane, a T-shaped build-
ing block (peptide T5) was designed to have a hydrophobic
dendron instead of the hydrophilic oligoether dendrons as in
peptides T3. The relative volume fraction of the hydropho-
bic dendron was adjusted to similar levels as peptide T3 so
as to drive efficient nanoring formation (Figure 3a).
Figure 2. Negative-stain TEM images of a) peptide T1 and b) peptide T2.
Bar=100 nm. Insets: CD spectra of the peptides (20 mm) in 20 mm KF.
Abscissa: wavelength [nm]; ordinate: [q]ꢁ10À3 [8cm2 dmolÀ1].
Peptide T5 was barely soluble in water owing to its in-
creased hydrophobicity, but it was clearly soluble in polar
organic solvents such as methanol. A CD spectrum of pep-
tide T5 in methanol shows that the building blocks do not
form well-defined secondary structures (Figure 3b). Howev-
er, a remarkable change in CD spectrum was observed
when peptide T5 was self-assembled in the presence of lipid
molecules (Figure 3b). For that experiment, peptide T5 was
mixed with lipid molecules (dioleoyl phosphatidylcholine or
DOPC) in methanol/chloroform, the solvents were dried,
and the dried film was sonicated in the presence of water
following standard liposome preparation protocols (peptide
T5/DOPC molar ratio=1:100). As shown in Figure 3b, pep-
tide T5 adopted a characteristic b-sheet conformation within
DOPC liposomes with an intense negative minimum of el-
lipticity at 216 nm, a strong positive maximum at 197 nm,
and a crossover point at 203 nm. This result is in line with a
previous report, which showed the potent ability of mem-
branes to promote the secondary structure of b-amyloid
peptides and a model hydrophobic hexapeptide.[34,35]
To further corroborate the result, we synthesized T-shape
peptide T2 (f=0.21), in which the bulkiness of a dendron
segment (first-generation triethylene glycol dendrimer) has
been reduced compared to that of nanoring-forming peptide
T3 (f=0.47). CD investigation shows that peptide T2 also
forms a b-sheet structure (Figure 2b, inset). Compared with
peptide T1, peptide T2 forms rather short fibrous objects
about 100 nm in length as a major population, together with
minor nanoring objects (Figure 2b). Similarly to peptide T1,
the fiber width (4.6 nm) corresponds well to the height of
peptide T2 (4.7 nm), thereby suggesting the formation of b-
ribbon structure. These results further emphasize that effec-
tive steric repulsion between the dendron segments is crucial
for efficient nanoring formation. In addition, weak steric re-
pulsion, although not sufficient to drive efficient nanoring
formation, is likely to interfere with 1D fiber growth, thus
making the self-assembly process prematurely terminate at
an early stage and resulting in the formation of rather short
fibers.[29,30] To gain further insight into nanoring formation,
we synthesized peptide T4 (f=0.76), which has a much bulk-
ier hydrophilic dendron (second-generation hexaethylene
glycol dendrimer) than that of nanoring-forming peptide T3.
The results showed that T4 does not adopt a clearly defined
To investigate whether or not the peptide T5 forms b-
barrel pores/channels within the lipid bilayer, we performed
a black lipid membrane experiment.[25,26] As illustrated in
Figure 3c, a typical current profile was obtained at an ap-
plied voltage of +40 mV across the membrane separating
1m KCl. This step-change behavior offers strong evidence of
a single-ion channel.[27] The current (I)/voltage (V) plot in
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from T5 is incorporated into
the lipid membrane and main-
tains their b-barrel structure so
it can transport ions through its
pore. Further insight into the
pore formation was revealed by
an ion-transport assay with a
small unilamellar vesicle (SUV)
entrapped with pH-sensitive
fluorophore HPTS (see the
Supporting
Information;
HPTS=8-hydroxypyrene-1,3,6-
trisulphonic acid). Following
peptide T5 addition at the indi-
cated time point (300 s), the
gradual ion transport across the
membrane was signaled by the
increase in relative fluorescence
that accompanied the rise in in-
travesicular pH.
The molecular arrangement
of the peptide segment in T5
produces a b barrel with hydro-
philic amino acid side chains at
the outer surface and hydro-
phobic side chains at the inner
surface, which might appear en-
thalpically unfavorable since
the hydrophobic inner surface
is exposed to the aqueous envi-
ronment. Although all the
above evidence supports the
formation of a b-barrel pore of
T5 in the lipid bilayer, we set
out to design more plausible
peptide models to further cor-
roborate the ionophore forma-
tion. The molecular arrange-
ment of the newly designed
peptides (T6) can drive the lo-
calization of hydrophilic side
chains to the inner surface and
hydrophobic side chains to the
outer surface of b barrels. As
Figure 3. T-shape peptides with hydrophobic dendrons. a) Structures. b) Open circle: a CD spectrum of T5
(20 mm in methanol). Closed circle: a CD spectrum of T5 in DOPC liposome (20 mm in water). c) Current pro-
file measured with and without T5 at +40 mV in 1m KCl. d) Single-channel I–V profile of conductance of T5
with a linear curve fit. e) Open circle: a CD spectrum of T6 (20 mm in methanol). Closed circle: a CD spectrum
of T6 in DOPC liposome (20 mm in water). Inset: Current profile measured with T6.
Figure 3d revealed an ohmic behavior of the ion channel
formed by the peptide b barrel, and from the slope, a single-
channel conductance of 263 pS was calculated for symmetri-
cal 1m KCl. The inner-channel diameter can be estimated
from the single-channel conductance by using Hilleꢂs equa-
tion. The corrected Hille diameter dHille ꢀ0.5 nm is smaller
than the inner diameter (3 nm) of the T3 nanoring. The
smaller diameter is likely to come from the further stabiliza-
tion of the b-sheet structure owing to mixing of hydrocar-
bons in dendrons and lipids, which is in line with previous
reports that membranes promote secondary-structure forma-
tion and self-association of hydrophobic peptides.[34–36]
Taken together, we can consider that a nanoring formed
shown in Figure 3e, the lipid membrane induced b-sheet for-
mation of otherwise unstructured peptide T6. The inset in
Figure 3e shows the typical current profile of peptide T6
across the membrane separating 3m and 0.2m KCl. This
step-change behavior is strong evidence of a single-ion chan-
nel.[37] The b-barrel peptide shows a single-channel current
of about 8.7 pA, which corresponds to an ion flux of 5.4ꢁ
107 ionssÀ1. Interestingly, it shows negative conductivity at
this asymmetric condition, which indicates that the pore pre-
fers chloride ion to potassium ion. The results suggest that
the amine residues located at the inner surface of the pore
play an important role in the anion transport, and the
degree of ionization between lysine and glutamic acid resi-
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Chem. Asian J. 2011, 6, 452 – 458

Designer Nanorings with Functional Cavities
20–150 mm KF. All sample solutions were incubated at least for 2 d at
room temperature before transferring to the grid.
dues is different. Therefore, all the data described above in-
dicate that T-shape building blocks can form b-barrel iono-
phores when properly designed. More in-depth study on the
precise structural requirements for b-barrel ionophore for-
mation should be subject of further investigation.
The geometry of the polyether group attached to the tyrosine side chain
for T3 was optimized by density functional calculations at the B3LYP/6-
31G(d) level. Electrostatic potentials were calculated based on the opti-
mized geometry, and the charges of atoms for the polyether group were
obtained through the RESP procedure.[38] The monomer of T3 was built
by linking the polyether group to the original b-sheet peptide (T1),
whereas the new parameters for the topologies and charges of the poly-
ether group were added to the FF96 force field with the XLEAP pro-
gram of Amber10. The T3 monomer was replicated and arranged to
make the 12-mer, 24-mer, and 40-mer of the toroidal pore structure with
the relative positions of the monomers adjusted to be similar to the struc-
ture of antiparallel b sheets. The initial structures were minimized with a
limited-memory Bryden–Fletcher–Goldfarb–Shanno quasi-Newton algo-
rithm and heated up to 500 K with the weak-coupling algorithm, whereas
the a-carbon atoms were fixed in space. After equilibration at 300 K
without the restraints, production molecular dynamic (MD) simulations
were performed. For all the computations, the concentration of salt was
set as 0.2m for neutralization and a modified generalized Born model
and the FF96 force field with modified hydrogen bonding radii were
used.[39]
Conclusion
We have shown how highly versatile peptide-based b-barrel
protein mimics with different external and internal surfaces
have been developed. The function of the b-barrel mimics
can be easily tuned by the simple change in dendron polari-
ty and/or b-sheet peptide configuration. Notably, our design
strategy enabled the control of the polarities of the inner
surface and outer surface of the b barrels at will. Consider-
ing that natural b-barrel proteins consist only of one long
polypeptide chain, it is remarkable that simple and relatively
small peptide building blocks can mimic natural b-barrel
protein folds through a pure noncovalent self-assembly pro-
cess. Hence, this study lays a foundation for the develop-
ment of versatile and biocompatible b-barrel protein mimics
that can displace the diverse biological functions of natural
b-barrel proteins with enhanced properties or with functions
unprecedented in nature.
Acknowledgements
M.L. thanks the National Creative Research Initiative Program of the
National Research Foundation (NRF) of Korea for a grant. Y.-b.L.
thanks the Translational Research Center for Protein Function Control,
Yonsei University for support through NRF grant no. 2010-0001933.
Experimental Section
Peptide was synthesized on Rink amide 4-methylbenzhydrylamine
(MBHA) resin with an Applied Biosystems model 433A peptide synthe-
sizer by using standard Fmoc SPPS protocols. Tyrosine derivatives were
coupled manually to resin-bound peptide with the O-benzotriazole-
N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) activation
method. Standard amino acid protecting groups were employed. For pep-
tide deprotection and cleavage, the resin was treated with cleavage cock-
tail (trifluoroacetic acid (TFA)/1,2-ethanedithiol/thioanisole: 95:2.5:2.5)
for 3 h and was triturated with tert-butyl methyl ether. The peptides were
purified by reverse-phase high-pressure liquid chromatography (HPLC;
water/acetonitrile with 0.1% TFA) on a C4 column (Vydac). The molec-
ular weight was confirmed with MALDI-TOF mass spectrometry. The
purity of the peptides was >95% as determined by analytical HPLC.
Concentration was determined spectrophotometrically in water/acetoni-
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