Structural requirements essential for elastin coacervation: Favorable spatial arrangements of valine ridges on the three-dimensional structure of elastin-derived polypeptide (VPGVG)n

Article abstract of DOI:10.1002/psc.1394

The elastin precursor tropoelastin possesses a number of polymeric peptides with repeating 3-9 mer sequences. One of these is the pentapeptide Val-Pro-Gly-Val-Gly (VPGVG) present in almost all animal species, and its polymer (VPGVG)n coacervates just as does tropoelastin. In the present study, in order to explore the structural requirements essential for coacervation, (VPGVG)n and its shortened repeat analogs (VPGV)n, (VPG)n, and (PGVG)n were synthesized and their structural properties were investigated. In our turbidity measurements, (VPGVG)n demonstrated complete reversible coacervation in agreement with previous findings. The Gly⁵-deleted polymer (VPGV)n also achieved self-association, though the onset of self-association occurred at a lower temperature. However, the dissociation of (VPGV)n upon temperature lowering was found to occur in a three-step process; the Val_i⁴-Val_i+1¹ structure arising in the VPGV polypeptide appeared to perturb the dissociation. No self-association was observed for (VPG)n or (PGVG)n repeats. Spectroscopic measurements by CD, FT-IR, and ¹H-NMR showed that the (VPGV)n and (VPG)n both assumed ordered structures similar to that of (VPGVG)n. These results demonstrated that VPGVG is a structural element essential to achieving the β-spiral structure required for self-association followed by coacervation, probably due to the ideal spatial arrangement of the hydrophobic Val residues.

Full text of DOI:10.1002/psc.1394

Research Article
Received: 19 February 2011
Revised: 30 May 2011
Accepted: 15 June 2011
Published online in Wiley Online Library: 14 September 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1394
Structural requirements essential for elastin
coacervation: favorable spatial arrangements
of valine ridges on the three-dimensional
structure of elastin-derived polypeptide
(VPGVG)n
Iori Maeda,a∗ Yoshiteru Fukumoto,a Takeru Nose,b
Yasuyuki Shimohigashi,b Takashi Nezu,c Yoshihiro Terada,d
Hiroaki Kodama,e Kozue Kaibaraf and Kouji Okamotoa
The elastin precursor tropoelastin possesses a number of polymeric peptides with repeating 3–9 mer sequences. One of these is
thepentapeptideVal-Pro-Gly-Val-Gly(VPGVG)presentinalmostallanimalspecies,anditspolymer(VPGVG)ncoacervatesjustas
does tropoelastin. In the present study, in order to explore the structural requirements essentialfor coacervation, (VPGVG)n and
its shortened repeat analogs (VPGV)n, (VPG)n, and (PGVG)n were synthesized and their structural properties were investigated.
In our turbidity measurements, (VPGVG)n demonstrated complete reversible coacervation in agreement with previous findings.
The Gly5-deleted polymer (VPGV)n also achieved self-association, though the onset of self-association occurred at a lower
temperature. However, the dissociation of (VPGV)n upon temperature lowering was found to occur in a three-step process; the
Vali4-Vali+11 structure arising in the VPGV polypeptide appeared to perturb the dissociation. No self-association was observed
for (VPG)n or (PGVG)n repeats. Spectroscopic measurements by CD, FT-IR, and 1H-NMR showed that the (VPGV)n and (VPG)n
both assumed ordered structures similar to that of (VPGVG)n. These results demonstrated that VPGVG is a structural element
essential to achieving the β-spiral structure required for self-association followed by coacervation, probably due to the ideal
c
spatial arrangement of the hydrophobic Val residues. Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: coacervation; elastin; repeat structure; self-association; spiral structure
Introduction
(Xxx Leu or Phe) are observed exclusively in bovine, porcine, and
human tropoelastins.
A common repeating sequence is the pentapeptide Val-Pro-
Gly-Val-Gly (VPGVG). This amino acid sequence is conserved in
Elastin is a major component of elastic tissues such as arterial
walls, lungs, and skin. Tropoelastin, a precursor protein of elastin
[1,2], assembles in a regular manner and is then cross-linked
by lysyl oxidase to form mature insoluble elastin in vivo. The
self-association of the tropoelastin molecule, which is called
coacervation, is the most important step in the process of
elastin biosynthesis. Tropoelastin forms a homogenous solution at
temperatures below 25 ◦C, but upon increasing the temperature
tobodytemperature(37 ◦C)orabove, thesolutionbecomesturbid
with coacervating droplets. Eventually, settling occurs to form a
dense viscoelastic coacervate layer. This feature is considered to
be related to the elastomeric function of elastin [3].
The amino acid sequence of tropoelastins varies among species
[4–8]. The typical structural feature of tropoelastin is the mutual
repeat of cross-linking regions and hydrophobic regions with
β-turns. The hydrophobic regions contain a distinct repetition
of peptide sequences. For instance, tripeptide Val-Pro-Gly and
heptapeptide Gly-Gly-Leu-Xxx-Pro-Gly-Val, where Xxx is Ala or
Val, are characteristic of chick tropoelastin. The tetrapeptide
Val-Pro-Gly-Xxx(Xxx AlaorGly), thehexapeptideVal-Gly-Val-Ala-
Pro-Gly and the nonapeptide Ala-Gly-Val-Pro-Gly-Xxx-Gly-Val-Gly
∗
Correspondence to: Iori Maeda, Department of Bioscience and Bioinformatics,
Kyushu Institute of Technology, 680-4, Kawazu, Iizuka, Fukuoka 820-8502,
Japan. E-mail: iori@bio.kyutech.ac.jp
a
b
c
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology,
Iizuka, Fukuoka 820-8502, Japan
Department of Chemistry, Faculty of Sciences, Kyushu University, Fukuoka
812-8581, Japan
Division of Dental Bioengineering, Department of Pathogenesis and Control of
Oral Diseases, Iwate Medical University School of Dentistry, Morioka 020-8505,
Japan
d
e
f
Department of Fixed Prosthodontics, Faculty of Dental Science, Kyushu
University, Fukuoka 812-8582, Japan
Department of Chemistry, Faculty of Science and Engineering, Saga University,
Saga 840-8502, Japan
Department of Biological Substances and Life Science, Faculty of Engineering,
Kyushu Kyoritsu University, Kitakyushu, Fukuoka 807-8585, Japan
c
J. Pept. Sci. 2011; 17: 735–743
Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.

MAEDA ET AL.
many animal species such as bovine, chick, porcine, and human,
and is considered to play a chief role in the elasticity of elastin. The
polypeptide (VPGVG)n has been reported to elicit a coacervation
property related to elasticity [9,10]. Urry et al. [11–13] carried out
conformational analyses of (VPGVG)n by CD and NMR techniques,
and showed that (VPGVG)n has a type II Pro2-Gly3 β-turn, resulting
in an ordered-state conformation called a β-spiral. However, the
structural requirements for formation of such a β-spiral structure
have never been reported, and thus the structural elements
intrinsic for self-association remain unknown. In order to evaluate
the contribution of the pentapeptide sequence to the property of
coacervation, a series of polymerized peptides (VPGVG)n, (VPGV)n,
(VPG)n, and (PGVG)n were synthesized in the present study.
We had specific reasons for choosing these peptides. The
truncated polytetrapeptide (VPGV)n was used to explore the role
of the Gly5 in the separation of the two Val residues, while the
properties of polytripeptide (VPG)n had the potential to reveal
why the VPGVG unit contains two residues of Val and Gly, in
the one case interrupted by Pro. Further, we hypothesized that
the polytetrapeptide (PGVG)n would reveal the importance of
the Val1-Pro2 interaction and the type II Pro2-Gly3 β-turn in the
Figure 1. Synthetic scheme of polytetrapeptide (VPGV)n, an elastin-
derived polymerized analog. To avoid racemization, the monomer
VVPG was polymerized; thus, the exact structural denotation should be
V-(VPGV)n-1-VPG.
1
(VPGVG)n repeat. By CD, FT-IR, and H-NMR methods, structural
analyses of all four peptides were performed together with a
coacervation assay.
It is difficult to examine by using spectroscopic analysis the
conformation of elastin-derived peptides in the coacervated state
because their self-assembly property can make the solution turbid
under this state. Therefore, different solution systems were used
to overcome the issue of turbidity in this spectroscopic study. A
report on the study of elastin-derived polypeptide conformation
showed the similarity of CD spectrum of polypentapeptide in TFE
with that of the coacervated polypentapeptide [9]. In addition, it is
wellknownthatTFEstabilizesstructureofpeptidesintoanordered
state in the CD measurements. Therefore, TFE was selected and
used as a solvent for CD and FT-IR measurements in this study,
like the other researchers did in their studies [14–16]. For NMR
measurements, we used DMSO as a solvent since DMSO has been
widely used for structural analysis of many peptides including
elastin-derived polypeptides [13,17], in addition to the ability of
DMSO to prevent the turbidity problem in the NMR study.
We here describe that the parent sequence VPGVG is essential
for both the construction of a three-dimensional structure and the
reversible self-association or coacervation.
The monomer unit of (VPGV)n was prepared as VVPG, with
Gly located at the C-terminal position to avoid racemization
in the polymerization reaction. Therefore, the exact structure
of polymerized tetrapeptide is denoted by V-(VPGV)n-1-VPG.
The purity of each monomer unit peptide, H-VPGVG-ONp, H-
VVPG-ONp, H-VPG-ONp, and H-PGVG-ONp, was verified by HPLC,
and the molecular weights of these peptides were confirmed
by MALDI-TOF-MS (Voyager-DE, Applied Biosystems, Foster City,
USA). Molecular weights of polymerized peptides were estimated
by SDS-PAGE.
The monomers of VPGVG, VPGV, VPG, and PGVG used for
1H-NMR analysis were synthesized by the solid-phase method
using the Fmoc strategy. For the coupling reaction, 0.45 M HBTU-
HOBt in DMF was used. Fmoc-amino acids, Fmoc-amino acid alko
resins, and other reagents for peptide synthesis were purchased
from Watanabe Chemical Ind. Ltd. (Hiroshima, Japan). All other
chemicals were purchased from Wako Pure Chemical Ind. Ltd.
(Osaka, Japan). Each coupling reaction was monitored by the
Kaiser test [22]. After completion of the peptide chain elongation,
a final cleavage was carried out by 95% TFA. The purity was
confirmed as described above. (VPGVG)2 was also prepared by
solid phase synthesis as described above.
Materials and Methods
Peptide Synthesis
All elastin-derived polypeptides were synthesized by the solution-
phase method using the Boc strategy. The condensation methods
used were the mixed-anhydride method [18] and the EDC/HOBt
method [19,20]. Deprotection of the Boc group was carried out in
4 M HCl/dioxane at 0 ◦C.
The polypentapeptide (VPGVG)n was synthesized according
to the polymerization technique reported previously [14,21].
Briefly, the monomer unit H-VPGVG-p-nitrophenyl ester (-ONp)
was first prepared by coupling between a peptide-free acid
and p-nitrophenol. The resulting H-VPGVG-ONp was polymerized
in concentrated DMSO solution for 2 weeks at 25 ◦C. The
polymerized peptide (VPGVG)n was collected by dialysis using a
3500 molecular-weight cut-off membrane. The synthetic scheme
of the novel polytetrapeptide (VPGV)n is shown in Figure 1.
Coacervation Measurement
The coacervation property was analyzed by measuring the
turbidity of the aqueous solution of each polypeptide. The
temperature-dependent turbidity was monitored and traced at
400 nm on a JASCO Ubest-50 spectrophotometer (JASCO Co.,
Tokyo, Japan). The concentration of the polypeptide solutions was
adjusted to 0.40 mg ml−1, and the measurements for turbidity
we◦re carried out in function of increasing temperature (from 5 to
60 C at 0.5 ◦C min−1) and then of decreasing temperature (from
60 to 5 ◦C at the same rate). These measurements were repeated
through several (at least two) temperature change cycles.
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STRUCTURAL ESSENTIALS FOR ELASTIN COACERVATION
CD Measurements
CD measurements were carried out in a 0.1 mm cuvette using
a JASCO J-725 spectropolarimeter (JASCO). Polypeptides were
dissolved in TFE (1.2 mM; estimated by using the molecular weight
of each peptide monomer unit) and measured at 25 ◦C. The
spectrum of the neat solvent was subtracted from the spectrum of
each sample. Measurements were carried out in the 190–280 nm
wavelength range.
FT-IR Measurements
FT-IR spectra of polypeptides in TFE (2.0 mg ml−1) were mea-
sured in 0.2 mm cuvettes on a JASCO FT/IR-615 spectrometer
(JASCO). Measurements were performed at 25 ◦C in the range
of 400–4000 cm−1 with 4 cm−1 resolution. Data from the amide
I region (1600–1800 cm−1) arising from the absorption caused
by C O stretch vibrations were collected, and the secondary
structures were determined by deconvolution (resolution en-
hancement) and the curve-fitting method [23–26]. In particular,
the amide I region was analyzed into individual structural compo-
nents with reference to the wavenumbers reported [27–29].
Figure 2. Coacervation profiles of elastin-derived polypeptides (VPGVG)n
(A) and (VPGV)n (B) in the measurement of turbidity at 400 nm. The
concentration of each polypeptide was 0.40 mg ml−1. Measurements
were carried out by increasing (so◦lid line) and decreasing (dotted line)
1H-NMR Measurements
the temperature between 5 and 60 C at a rate of 0.5 ◦C min−1
.
1H-NMR spectra of peptides were recorded on either a Bruker
AM-400 spectrometer (400 MHz) or a JEOL JNM-A500 (500 MHz) at
298 K. Chemical shifts were determined using TMS as an internal
standard. All peptides were dissolved in DMSO-d6 (5 mg ml−1),
and the signals were assigned by 2D phase-sensitive double
quantum-filtered COSY (DQF-COSY).
around 37 ◦C, the solution became suddenly turbid, and the tur-
bidity reached a maximal level by 60 ◦C. This turbidization simply
indicates the onset of coacervation in that temperature range.
TheturbiditycurveriseswithasharpslopeasshowninFigure 2A.
When the solution was allowed to cool down, the turbidity curve
traced exactly the same path. Thus, the curves of the elevation
anddeclineoverlappedcompletely(Figure 2A). Thismeasurement
was repeated several times, and the curves always overlapped.
On the other hand, such an overlap was not observed in the case
of the polytetrapeptide (VPGV)n repeat. As shown in Figure 2B,
the increase in turbidity occurred at 25–50 ◦C, i.e. at a much lower
temperature than that of (VPGVG)n. This is a very prominent and
definite difference; the temperature difference for the start of
the ascent was approximately 10 ◦C lower than that of (VPGVG)n
(Figure 2A and B). Another important difference was the steeper
slope (ca 1.7-fold) of the ascent by (VPGV)n. When the slopes of
the elevations were calculated, that of (VPGV)n was 0.24/ ◦C, while
that of (VPGVG)n was 0.14/ ◦C. Since the molecular size of these
polypeptides is almost same, these results clearly indicate that the
formation of a highly ordered structure of (VPGV)n proceeds much
more readily than that of (VPGVG)n.
This process of formation of the highly ordered structure can be
correlated to the recorded course of decline shown in Figure 2B.
The dissociation process consists of at least three distinct steps
over a rather broad temperature range (60–5 ◦C). The first step
(60–42 ◦C) was very slow (0.0003/ ◦C), but relatively short. The
second step (42–20 ◦C) was moderately slow, at the rate of
0.02/ ◦C. By contrast, the third step (20–5 ◦C) was very fast and
considerably steep (0.18/ ◦C). It should be noted that this last
steep of dissociation is just like the decline observed for (VPGVG)n.
These two descent curves appear very similar, based on the similar
slopes.
Results
Polymer Preparation and Their Average Polymerization
Degree
Elastin is distributed in elastic tissues to impart elasticity to the
tissues, and the pentapeptide VPGVG takes a major role in these
elastic functions. The repetition of (VPGVG)n is approximately four
to ten times, varying according to species [4–7]. The molecular
weights of monomeric peptides used in the polymerization
reaction were confirmed by MALDI-TOF-MS as follows; H-VPGVG-
ONp: 549.20 (549.57 calcd), H-VVPG-ONp: 491.62 (492.52 calc),
H-VPG-ONp: 393.10 (393.39 calcd), and H-PGVG-ONp: 449.20
(450.44 calcd). The molecular weight of synthetic (VPGVG)n was
measured by SDS-PAGE, and its average molecular weight was
estimatedtobeapproximately60,000. Giventhisvalue, thedegree
of polymerization was calculated to be about 150, much larger
than that in native tropoelastin. The polymerization degrees of
other polymers, (VPGV)n, (VPG)n, and (PGVG)n, were estimated
by the same method and were approximately 170, 120, and 190,
respectively.
Coacervation Property of (VPGVG)n and Its Analogs
Figure 2 shows the temperature-dependent coacervation profiles
of the elastin-derived polypentapeptide and its analogs. It is evi-
dent that the (VPGVG)n repeat possesses a distinct coacervation
property (Figure 2A). It exhibited completely reversible coacerva-
tion as reported previously [21]. When the temperature was raised
continuously up to 60 ◦C, starting from 5 ◦C, no turbidity was
observed until 35 ◦C. Immediately after reaching a temperature
In contrast, no coacervation was observed for (VPG)n or (PGVG)n
(data not shown). Absolutely no turbidity was observed when the
temperature was raised to 60 ◦C. These results suggest that both
the Val1 and Val4-Gly5 residues are essential for coacervation. Also,
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MAEDA ET AL.
4
2
Table 1. Characteristic frequencies in amide I region of curve-fitted
FT-IR spectra of polypeptides
(VPGVG)n
Band
frequency
Relative
intensity (%)
(cm−1
)
Assignment
0
Polypeptide
(VPGVG)n
1615
1633
1656
1680
20
28
28
24
β-Aggregation
β-Sheet structure
Random coil
-2
-4
-6
-8
-10
β-Turn structure
(VPGV)n
(VPG)n
1615
1633
1656
1680
14
32
26
27
β-Aggregation
β-Sheet structure
Random coil
(VPGV)n
β-Turn structure
(PGVG)n
200 210
(VPG)n
230
1615
1633
1656
1680
16
34
27
23
β-Aggregation
β-Sheet structure
Random coil
190
220
240
250
Wavelength (nm)
β-Turn structure
Figure 3. CD spectra of elastin-derived polypeptides (VPGVG)n (black),
(VPGV)n (red), (VPG)n (blue), and (PGVG)n (green). All polypeptides were
dissolved in TFE at a concentration of 1.2 mM (estimated by the molecular
weight of each unit), and measurements were carried out at 25 ◦C.
(PGVG)n
1605
1641
32
32
Unknown
Random coil, β-turn
and β-sheet
1672
36
β-Turn structure
the Gly5 residue appears to be important for the reversibility or
equilibrium in coacervation.
ConfirmationofthePresenceofCarbonylGroupintheβ-Spiral
Structure by FT-IR
CD Measurements to Prove the Presence of an Ordered
Structure with β-Turn
Figure 4 shows the FT-IR spectra of elastin-derived polymeric
peptides in the range of 1500–1800 cm−1. In this region, the
FT-IR spectra show the presence of absorption bands brought
about by a series of amide I carbonyl groups (C O). Polypeptides
(VPGVG)n, (VPGV)n, and (VPG)n exhibited similar spectra, and each
curve was decomposed into four components at 1615, 1633, 1656,
and 1680 cm−1 in the same pattern (Figure 4A–C and Table 1).
The band of 1615 cm−1 is considered to indicate the existence
of the β-aggregation [28,29]. The absorption band at 1633 cm−1
is attributed to the amide C O groups involved in the hydrogen
bonding of the β-sheet structure [27], and intense absorptions
were observed for all of the VPG-containing polymeric peptides.
The band at 1656 cm−1 was attributed to the amide C O groups
of the random coil structure [28,29]. This band was observed in
all the repeats, namely, (VPGVG)n, (VPGV)n, and (VPG)n, and thus
these polypeptides must contain such non-hydrogen bonding
All the CD spectra were measured at 25 ◦C, and thus under the
solution condition with no coacervation or aggregation. It was
previously reported that (VPGVG)n exhibits a β-spiral structure, in
whichPro2-Gly3 formsatypeIIβ-turnatthecornerofthespiral[30].
Indeed, in the present study, the spectrum of (VPGVG)n exhibited
a type II β-turn structure with a distinct negative peak at 224 nm
together with a positive peak around 208 nm (Figure 3), just as
reported by Urry et al. [11]. This profile is certainly a characteristic
of the β-spiral structure adopted by elastic polymerized peptides.
The CD spectrum of (VPGV)n was found to be very similar to that
of (VPGVG)n, although a slight blue-shift (ca 5 nm) was observed
for the positive peak. The total profile indicated the presence of
a type II β-turn-like structure as shown by (VPGVG)n. Since the
positive Cotton effect with a peak at 203 nm was judged to be
broad compared to that of (VPGVG)n, it is possible that the spiral
structure with a β-turn is slightly modified in the polytetrapeptide.
When the spectrum of (VPG)n was compared with those of
(VPGVG)n and (VPGV)n, it was clear that its CD profile has a
larger negative band at 221 nm, with a maximum blue-shifted
approximately by 3 nm compared to the negative maxima of
the other two spectra (Figure 3). Since there is a positive band
at 195 nm (ca 13 nm blue-shifted from that of (VPGVG)n), the
spectrum shows the predominant presence of a β-structure,
probably with consecutive type II β-turns.
C
O groups in an ordered structure, that is, in a β-spiral structure.
In fact, this band is considered to be attributed to Pro2-C O and
Gly3-C O. The band that is attributed to the amide C O groups
in a β-turn emerged at 1680 cm−1 [27]. Since the absorption
band was elicited by the hydrogen bond that stabilizes the β-turn
structure, it was supposed that (VPGVG)n, (VPGV)n, and (VPG)n
share a β-turn, where Vali1-C O is very likely a hydrogen acceptor
in the hydrogen bond with Vali4-NH or Vali+14-NH. Judging from
the amounts of each decomposed component by deconvolution
and the curve-fitting method (Table 1), it was suggested that these
three polypeptides contain a similar ordered structure as indicated
in the CD measurement. In contrast, the (PGVG)n exhibited a
different spectrum from those of the other three polypeptides
(Figure 4D), and it decomposed into three different absorption
bands at around 1605 (unknown), 1641 (random coil, β-turn, and
β-sheet), and 1672 cm−1 (β-turn).
On the other hand, the spectrum of (PGVG)n was found to be
distinctly different from the spectra of any of the other polymeric
peptides measured in this study. The curve suggests the presence
of an unordered structure characterized by a strong negative band
below 200 nm. All the results from CD spectra suggest that the
two polypeptides (VPGV)n and (VPG)n form ordered structures as
(VPGVG)n does, whereas (PGVG)n does not form such an ordered
structure.
c
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STRUCTURAL ESSENTIALS FOR ELASTIN COACERVATION
B
A
0.10
1633
1656
1633
1656
0.10
1615
1680
1680
1615
0
0
1600
1700
1600
1700
Wavenumber (cm-1)
Wavenumber (cm-1)
C
D
0.20
0.10
1633
1641
1672
1656
1680
1615
1605
0
0
1700
1600
1700
1600
Wavenumber (cm-1)
Wavenumber (cm-1)
Figure 4. FT-IR spectra of elastin-derived polypeptides (VPGVG)n (A), (VPGV)n (B), (VPG)n (C), and (PGVG)n (D). All polypeptides were dissolved in TFE
(2.0 mg ml−1), and measurements were carried out at 25 ◦C in the 400–4000 cm−1 wavenumber range.
1H-NMR Analysis of Val-γ -Methyl Groups in an Ordered
Structure
NMR-proton signals of methyl groups attached to the β-carbon.
The NMR signals of these methyl (CH3) groups were recorded
at the range of 0.7–1.1 ppm. It was immediately apparent that
the signals of Val1 (doublet–doublet; dd) and Val4 (dd) were
completely separate: i.e. 0.84 ppm for the CH3 of Val4 in G3V4G5
and 0.98 ppm for the CH3 of Val1 in the N-terminal V1P2 (Figure 5,
monomers).
In the present study, we measured the NMR spectra of mono-,
di-, and polymeric derivatives of the pentapeptide sequence of
VPGVG. As shown in Figure 5, for example, very finely resolved
resonance signals were observed for all of these derivatives. It was
very surprising to attain well-resolved spectra for the polymeric
(VPGVG)n repeat. The methyl protons of Val1 and Val4 were clearly
separated and finely resolved (Figure 5). Also, the amide N–H
protons of Val1, Gly3, Val4, and Gly5 were distinctly separated at
the baseline and assigned to their respective signals (data not
shown). These results definitely indicate that (VPGVG)n assumes
an extremely highly ordered structure. Such a clear separation of
the methyl and amide protons was also identified in the spectra of
(VPGV)n and (VPG)n.
Inthe(VPGVG)nrepeat, inwhichthemonomericpeptideVPGVG
is repeated over and over again, two different kinds of Val residue
emerge: i.e. Val1 in the G5V1P2 sequence and Val4 in the G3V4G5.
TheexceptionisonlytheN-terminalValresidue, whichistruncated
directly without the preceding Gly5 residue. Given that these Val1
and Val4 residues are present in a strictly ordered structure,
they should be grouped distinctively with different structural
circumstances. This was indeed demonstrated in the 1H-NMR
spectroscopy as shown in Figures 5 and 6. Val is characterized
by its side chain isopropyl group, and it can be observed by the
Since the N-terminal Val1 is an exception in the polymeric
(VPGVG)n repeat, we then measured the dimeric (VPGVG)2
=V 1PGV 4GV 1’PGV 4’
G
1
(
) to investigate the chemical shift of Val in
ꢁ
the GVP sequence. As shown in Figure 6, Val1 in GV1 P was easily
distinguished. The signal (dd) of this methyl group emerged at
the intermediate resonance site of 0.88 ppm. Thus, as seen clearly
in the spectrum of the (VPGVG)n polymer (Figures 5 and 6), there
are two major Val-CH3 signals: namely, the doublet–doublet at
0.84 ppm (for Val4-CH3) and the doublet–doublet at 0.88 ppm (for
Val1-CH3), although the doublets completely overlap at 0.85 ppm.
This is certainly a demonstration that all the Val1 residues are
in the same structural circumstances as the Val4 residues. Thus,
(VPGVG)n must have an extremely ordered structure, namely the
β-spiral structure.
In the case of the tetrapeptide VPGV and its polymer, the signals
of Val1 (dd) and Val4 (dd) were found to be much more clearly
separated (Figure 5): i.e. the CH3 of Val4 in G3V4V1 at 0.78 ppm and
the CH3 of Val1 in V4V1P2 at 0.88 ppm. There is no overlapping
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J. Pept. Sci. 2011; 17: 735–743 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

MAEDA ET AL.
Figure 5. 1H-NMR spectra of Val-γ CH3 proton signals of the monomer and
polymer of VPGVG together with a series of their shortened derivatives,
VPGV,VPG,andPGVG.AllpeptidesweredissolvedinDMSO-d6 (5 mg ml−1).
of signals, and the signals of the doublet–doublet are distinct,
indicating that both Val residues are placed in an extremely
ordered and restricted conformation.
VPG anditspolymer(VPG)nexhibitedverysimpleprotonsignals
of Val (dd) at 0.98 and 0.87 ppm, respectively (Figure 5). Since this
tripeptide repeat possesses only a single type of Val in the G3V1P2
sequence, it is quite reasonable to assume that (VPG)n also adopts
a highly ordered uniform structure. In contrast, the single-Val-
containing (PGVG)n repeat exhibited a rather complicated signal
pattern as shown in Figure 5. Its monomer showed a very fine dd
signal at 0.87 ppm. In spite of the presence of a single class of
Val in the GVG sequence, the NMR profile suggests that (PGVG)n
possesses at least two different orientations of Val residues, and
thus this repeat adopts a conformation completely different from
those of the other repeats using in this study.
The number of repeats in the polymer (VPGVG)n sample was
estimated to be approximately 150 as mentioned above. Those
of the (VPGV)n and (VPG)n samples were estimated as 170
and 120, respectively. In spite of the extremely high degree of
polymerization, their 1H-NMR spectra were finely resolved. These
results clearly indicate that the polymers (VPGVG)n, (VPGV)n, and
(VPG)n are probably in a uniformly restricted conformation, that
is, a β-spiral structure, a β-spiral-like structure, and an ordered
structure possibly a β-structure, respectively.
Figure 6. 1H-NMR spectra of Val-γ CH3 proton signals of pentapeptide
repeats VPGVG (A), (VPGVG)2 (B), and (VPGVG)n (C). All peptides were
dissolved in DMSO-d6 (5 mg ml−1).
hydrophobic interactions between Val residues as well. The chief
structuralcharacteristicofthepentapeptiderepeat(V1P2G3V4G5)n
is the two line-ups of hydrophobic Val residues. Contact of the
Val4-Val4 side chain between intermolecular polypeptide is im-
portant for proper intermolecular hydrophobic association in
(V1P2G3V4G5)n [30]. It was reported that there are two basic
elements for the β-spiral structure of (V1P2G3V4G5)n [31–33]. One
is the presence of a type II Pro2-Gly3 β-turn as the dominant
secondary structure [34,35], and the other is the utilization and
optimization of intramolecular hydrophobic contacts resulting in
a loose helical structure. It was also described that β-turns are
suspended by the Val4-Gly5-Val1 peptide segment with large-
amplitude, low-frequency rocking motions of the (V1P2G3V4G5)n
moleculeitself.Thesecharacteristicswereconfirmedinthepresent
study by a series of spectroscopic measurements, i.e. CD, FT-IR and
1H-NMR. The type II Pro2-Gly3 β-turn is stabilized by the hydro-
gen bond between Val1-CO and Val4-NH (Figure 7A), and also
by the side chain–side chain interaction between Val1-γ CH3 and
Discussion
The pentapeptide repeat (V1P2G3V4G5)n exhibits a coacerva-
tion property characterized by reversible association/dissociation.
Clearly, the self-association is initiated by not only the intramolec-
ular Val1-Pro2 side chain association but also by the intermolecular
c
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STRUCTURAL ESSENTIALS FOR ELASTIN COACERVATION
Figure 7. Schematic diagram of the backbone structure of polymeric peptides (VPGVG)n (A), (VPGV)n (B), and (VPG)n (C). Wavy arrows depict the hydrogen
bonding between the peptide bonds: i.e. A and B, between the amide-carbonyl group of Val1 and the amide-imino group of Val4; and C, between the
amide-carbonyl group of Val1 and the amide-imino group of the next Val1. Between the two arrows, there are seven linking atoms in (VPGVG)n (A), four
in (VPGV)n (B), and one in (VPG)n (C).
Figure 8. Illustration of a type II β-turn structure putative for polymeric peptides (VPGVG)n (A), (VPGV)n (B), and (VPG)n (C). The first peptide unit structures
are depicted, and the curving arrows show the direction of the spiral structure.
Pro2-βCH2 as shown in Figure 8A. The hydrogen bond between
Val1-CO and Val4-NH brings about a ring structure with ten atoms,
and the resulting β-turn is repeated over and over by inserting the
Val4-Gly5-Val1 peptide segment (Figure 7A). The β-turn hydrogen
bonds are separated by seven atoms in the Val4-Gly5-Val1 suspen-
sion as shown in Figure 7A. Collectively, the β-spiral structure of
(V1P2G3V4G5)n is very similar to 310 helix structure.
residues would be placed on the same ridge. Meanwhile, all the
Val4 residues are located on a ridge just adjacent to it; hence, the
1
consecutive Vali4-Vali+1 residues create a large ridge consisting
of the four methyl groups.
1
For the Vali4-Vali+1 widened row, we denote the line-up with
four γ CH3 groups as the face and the line-up with one or twoγ CH3
groupsastheedge.Inthisscheme,theedge-to-edgeintermolecular
interaction would require the least energy and be the weakest
among the possible interactions of the tetrapeptide repeat
(V1P2G3V4)n. Ontheotherhand, the face-to-face interactionwould
requirethemostenergyandwouldalsobethestrongest.Theedge-
to-faceinteractionwouldbetheintermediateoftheseinteractions.
Thus, in the interactions between the tetrapeptide repeat
(V1P2G3V4)n molecules, three different mechanisms, namely edge-
to-edge, edge-to-face, and face-to-face interactions, occur in this
order with increasing temperature. The first mechanism would
be much faster and larger than the latter two mechanisms. It
is likely that these mechanisms take place at the same time
for the self-association of the Gly5-deleted polymer (V1P2G3V4)n.
In this study, the tetrapeptide repeat (V1P2G3V4)n was found
to adopt a β-spiral-like structure. This polymer possesses a type II
Pro2-Gly3 β-turn stabilized by a hydrogen bond between Val1-CO
and Val4-NH. The β-turn with the hydrogen bond also makes a
ten-atom ring, which is separated from the next ring by only four
atoms, namely, Cα4-C( O)-N(-H)-Cα1, in the Val4-Val1 residues
(Figures 7B and 8B). The shortening of the suspension is attributed
to the deletion of Gly5, and this deletion causes the spiral structure
to tighten. Further, the deletion of Gly5 causes the Val residues
to be adjacent to each other, resulting in the formation of a very
1
rigid spiral structure. Since the H-NMR measurement indicated
that all of the Val1 residues are in the same circumstance, these
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J. Pept. Sci. 2011; 17: 735–743 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

MAEDA ET AL.
Indeed, this appeared to happen in our turbidity measurement
experimentsshowninFigure 2B. Thetetrapeptiderepeatachieved
a self-association at a much lower temperature range compared
with that by the pentapeptide repeat (V1P2G3V4G5)n (Figure 2).
After achievement of self-association, dissociation should occur
with the decrease of temperature. It is reasonable to assume
that the dissociation would occur in the reverse energy order of
the mechanisms of the three self-association interactions; i.e. the
order edge-to-edge, edge-to-face, and face-to-face is reversed. It
should be noted that three-step dissociation was in fact observed
in the lower temperature range (Figure 2B). The dissociation of
the face-to-face interaction occurred first, and it was the slowest
and the smallest in terms of numbers of the interactions among
(C O) of Val1 and the amide-imine group (N-H) of Xaa4 in VPGX;
then, the polymeric peptide repeat builds a β-spiral structure. The
β-spiral structure creates a line-up of Val1 residues in addition
to a line-up of Val4 residues to guarantee the intermolecular
interaction followed by self-association. This additional line-up of
the Val1 residues is not directly adjacent to the line-up of the Val4
residues; the two line-ups of Val4 and Val1 residues are separated
from each other by one or two amino acids.
All of these conditions are realized by the wild-type pentapep-
tide (VPGVG)n repeat, but not by its shortened analogs (VPGV)n,
(VPG)n, and (PGVG)n. All the results in this study demonstrated
that VPGVG is a structural element essential to achieving the β-
spiral structure that is superlative for self-association followed by
coacervation, due to the ideal spatial arrangement of hydrophobic
Val residues. In conclusion, only the wild-type (VPGVG)n repeat
can play an intrinsic essential role in the elastomeric function of
elastin.
There are some reports [29,38] contradicting Urry’s studies
that (VPGVG)n has β-spiral structure. Although it was suggested
that (VPGVG)n has β-turn structure, the clear mechanism of
coacervation of elastin-derived peptide was not shown definitely
in their reports. Furthermore, it is difficult to explain elastin’s
completely reversible coacervation property by considering
only water molecule adaptation and the β-strand structure of
polypeptides. As shown in Urry’s recent report [39,40], more
detailed structural analysis is needed for determination of
the structural change of elastin-derived peptides affected by
temperature. A slight modification of his model as presented
in this study may explain the precise dynamic mechanism of
coacervation.
1
the three mechanisms. Collectively, the consecutive Vali4-Vali+1
structurearisinginthe(V1P2G3V4)nrepeatappearedtoperturbthe
dissociation by the additional two mechanisms, the edge-to-face
and edge-to-edge.
The tripeptide repeat (V1P2G3)n also assumes an ordered
structure with a type II Pro2-Gly3 β-turn stabilized by a hydrogen
bond between Val1-CO and the NH of the next residue (denoted
ꢁ
ꢁ
as Val1 hereafter) Val1 -NH (Figure 7C). The ten-atom β-turnꢁ ring
structures are separated by only a single atom, namely, Val1 -Cα,
in the Val1-Val1 residues as shown in Figure 8C. The deletion of
ꢁ
Val4-Gly5 makes the spiral structure of (V1P2G3)n much tighter
than that of (V1P2G3V4)n (Figure 8). CD spectra of (V1P2G3)n reveal
a type II β-bend structure under concentrated conditions even in
water at room temperature as reported before [36], suggesting
that (V1P2G3)n tends to have ordered rigid structure. Because
of the deletion of Val4-Gly5, there are successive, uninterrupted
β-turns, and this puts the Val residues on a single ridge only.
Although each molecule is able to produce something like a fiber
by continuous associations, it is impossible to make an assembly
owing to the presence of only a single line-up of Val residues. This
is compatible with the result of the turbidity experiment. Since the
tripeptide repeat (V1P2G3)n is a component of chick tropoelastin
as mentioned before, this tripeptide repeat might have a different
role in the elastomeric function of elastin.
Acknowledgement
We thank to Mr. Masumi Kunisue (Center for Instrumental Analysis,
Kyushu Institute of Technology) for technical support with the
1H-NMR measurements.
In the case of the tetrapeptide repeat (P2G3V4G5)n, we did not
find any evidence that this polymer adopts an ordered structure.
This polypeptide can be regarded as a mutant of for (V1P2G3V4)n,
with G5 in (P2G3V4G5)n replacing V1 in (V1P2G3V4)n when
considering the continuous sequence of polymer. Interestingly,
this substitution caused a big difference in coacervation property
of each polypeptide. This result clearly indicated that this big
difference in coacervation property is caused by losing the
sequence, VPGV, whichisnecessaryforthecoarcervationproperty.
As mentioned above, the presence of V1P2G3 is important to
stabilize the type II Pro2-Gly3 β-turn structure by the hydrogen
bondtogetherwiththesidechain–sidechaininteractionbetween
Val1 and Pro2. Instead, the (P2G3V4G5)n repeat possesses G5P2G3,
which lacks the side chain–side chain interaction of Pro2 because
Gly5 has no side chain. A NOE study has demonstrated the
importance of Val1 and Pro2 by an increase in the intramolecular
hydrophobic association of the γ CH3 of Val and the δCH2 of Pro at
increasing temperatures [37].
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