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The structures of elastin and their function

Article in Biochimie · November 1999

DOI: 10.1016/S0300-9084(99)00221-7 · Source: PubMed

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Biochimie 81 (1999) 981-994
© 1999 Soci6t6 fram;aise de biochimie et biologie mol6culaire / t~ditions scientifiques et m6dicales Elsevier SAS. All rights reserved

T h e s t r u c t u r e s of elastins a n d their f u n c t i o n

Laurent Debelle, Alain J.E Alix*

Laboratoire de Spectroscopies et Structures Biomol~culaires, Universit~ de Reims-Champagne Ardenne, Facult~ des Sciences,
Moulin de La Housse, B.P. 1039, 51687 Reims cedex 2, France
(Received 5 January 1999; accepted 7 June 1999)

A b s t r a c t - Elastin structures and their significance towards elastic recoil properties have been reviewed. Starting from the initial
hypothesis that elastin conformation is conditioned by that of its monomer, the structure of tropoelastin was first described using
theoretical and experimental methods and a [3 class folding type was evidenced for the isolated unbound tropoelastin molecules. The
structure of elastin in the solid state was consistent with that of its monomer and consequently, fibrous elastin appeared constituted
of globular tropoelastin molecules. Finally, theoretical and experimental considerations have led us to the conclusion that the
functional form of the elastomer, water swollen elastin, could be a triphasic system comprising the protein chains, hydration water and
solvent water. Following this description, the dynamic structural equilibria occurring within elastin hydrophobic domains and the
plastisizing effect of water could explain elastin elasticity, in keeping with a classical entropic mechanism. © 1999 Soci6t6 frangaise
de biochimie et biologie mol6culaire / Editions scientifiques et m6dicales Elsevier SAS
elastin / secondary structures / elasticity

1. Introduction cular dichroism (CD) [6-15], Fourier transform infrared

Elastin [1] is the extracellular matrix protein respon-
sible for the resilience of tissues such as skin, arteries and
lung [2]. It is an insoluble, hydrophobic and extensively
cross-linked protein forming fibers which are present in
variable amounts depending on the tissue. Although it has
been involved in numerous biological activities, elastin's
function is restricted to elasticity.
Elastin elasticity is of entropic origin [3], but despite
several decades of constant efforts, this phenomenon is not
fully understood yet [4]. This is mainly due to the fact that
the structure of the elastin network is still not fully
understood, and, as a consequence, the structure-elasticity
relationships remain unclear.
Since the pioneering work of Partridge and co-
workers [5] which revealed that the polypeptide chains of
the constitutive tropoelastin monomers were joined by the
specific desmosine cross-links, numerous structural stud-
ies dealing with purified or solubilized elastin have been
reported. However, the results gathered using either cir-
* Correspondence and reprints
Abbreviations: BTE, bovine tropoelastin; CTE, chick tropoelas-
tin; HTE, human tropoelastin; MTE, mouse tropoelastin; RTE,
rat tropoelastin; STE, sheep tropoelastin; BE, bovine elastin; HE,
human elastin; RE, rat elastin; BKE, bovine ~-elastin; GOR,
prediction method of Gamier, Osguthorpe and Robson; CD,
circular dichroism; FTIR, Fourier transform infrared; NIR VrR,
near infrared Fourier transform Raman; NMR, nuclear magnetic
resonance; ct, a-helix; [3, ~-sheet; U, undefined conformation
(turns + random coil).
(FTIR) [6, 16, 17], classical Raman [18, 19], fluores-
cence [20, 21] and nuclear magnetic resonance (NMR)
spectroscopies [22] or X-ray diffraction [23] methods
were severely limited because of the extreme insolubility
of the mature cross-linked elastin and the coacervation
property of its derivatives [24-26]. In short, it was shown
that some level of order existed within the polymer
conformation and that its chains were highly mobile.
Thus, no definitive conclusions could be reached except
that elastin was largely disordered. Historically, the 'oiled
coil' molecular model [27] constituted the first attempt to
associate specific structures to the elastic function. In this
model, loop structures (the oiled coil) were assigned to the
large hydrophobic regions responsible for the elasticity of
the polymer while the cross-linking domains were de-
scribed as a-helical ones, a commonly accepted view.
Although partial tropoelastin sequences were already at
hand [28], the complete sequencing of tropoelastin from
several species [29-34] was a considerable breakthrough
as it allowed to individuate the various regions participat-
ing in elastin structure and function. The cross-linking
domains were identified as being either alanine-rich
(termed KA) or proline-rich (termed KP) regions compris-
ing either two or three lysyl residues. Likewise, the very
hydrophobic regions responsible for elasfin elasticity ap-
peared to be formed of extensive, overlapping repetitions
of X G G X G and XPGXG peptides, where X = V, A, L or
I. Owing to the difficulties encountered in the study of
purified elastin, these peptides represented an interesting
alternative and, consequently, a considerable effort was
982
devoted to the study of their structures and dynamics
using either experimental [35-45] or theoretical [46-51]
methods.
One of the most important achievements of those
studies was the demonstration that both type§ possessed
the ability to form type II [5-turns. However, the arrange-
ment and dynamic behavior of these structures varied
depending on the type of considered peptide. Urry [39, 52]
suggested a regular arrangement, the [5-spiral, for the
XPGXG type polypeptides. According to this model, the
entropic elasticity would originate from librational mo-
tions [52]. This proposal is still discussed but we must
underline that it holds for rather long stretches of XPGXG
repeats [39] and that in tropoelastin, such repeats occur
mostly in the sequence encoded by exon 18 and 24 [53].
The structures of XGGXG peptides and polypeptides have
been extensively studied by Tamburro and co-workers
(see [4], and references cited therein) and, in contrast to
Urry's model, they underlined that the [5-turns occurring
in these sequences should be very labile and dynamic
ones. Thus, these structures would greatly contribute to
the entropy of elastin and elasticity could be explained by
a classical mechanism [3].
The two models cited above aimed at the elucidation of
elastin structure-elasticity relationships but both were
devised with the assumption that molecular features ob-
served in these elastin-like synthetic peptides were rel-
evant for those occurring in elastin. This straightforward
inference is not necessarily true and, consequently, the
question of the relevance of the synthetic models was
raised.
In order to address this point, our group used another
approach. First, we took advantages of the known se-
quences of the bovine (BTE) and human (HTE) tro-
poelastins and their secondary structures were pre-
dicted [54]. Second, the secondary structures of BTE were
characterized experimentally and their percentage con-
tents were used to adjust the former predictions. That way,
a pseudo-3D molecular model of tropoelastin could be
proposed [55]. Finally, bovine elastin was purified and its
secondary structures were analyzed by optical spec-
troscopies [56]. This permitted us to demonstrate that the
architectural model of elastin was consistent with the
liquid drop construction of Weis-Fogh and Andersen [57],
i.e., fibrous elastin is constituted of globular monomers.
This architectural model has been confirmed by atomic
force microscopy [58]. An important point of our results
was the identification of substantial amounts of irregular
and distorted [5-structures within elastin conforma-
tion [56]. Additionally, they stressed the importance of
hydration water as a determinant factor of the conforma-
tional entropy of the polymer [59].
Dry elastin is brittle while water-swollen elastin is
highly elastic [60, 61]. This is explained by the fact that
water acts as a plasticizer for the polymer [62, 63], i.e., it
considerably raises the conformational entropy of the
Debelle and Alix
protein. This point is a fundamental aspect of elastin
elasticity but its explanation in structural terms is unclear.
Indeed, if it is now well established that the structures of
elastin are mobile and influenced by the presence of
water [4], how and where it interacts with the polypeptidic
chains at the molecular level, can only be guessed [4, 62].
The aim of this review is to propose a molecular
description of elastin, and, to attempt to explain its
elasticity. For this purpose, our previous results have been
updated and reconsidered in keeping with the considerable
literature at hand concerning synthetic elastin-like pep-
tides. That way, the molecular model we propose here
provides a synthetic description of the current knowledge
on elastins structures. Additionally, its structure-elasticity
relationships are discussed underlining the probable in-
volvement of elastin structures in its entropic elasticity.
2. The structure of tropoelastin
The primary transcripts of the elastin gene are subject
to extensive alternative splicing [30-33] and consequently
various tropoelastin isoforrns are translated. For simplici-
ty's sake, we will always refer to the full sequence of
tropoelastins, while the hydroxylation of some of its prolyl
residues (about 1% Hyp in elastin)[26] will not be
considered unless stated otherwise.
2.1. Alignment of tropoelastin sequences
The full sequences of chick (CTE)[29], human
(HTE) [30], bovine (BTE)[31], rat (RTE)[32], sheep
(STE) [33], and mouse (MTE)[34] tropoelastins have
been aligned using exon boundaries constraints (figure 1).
Tropoelastins shared considerable sequence homology,
more than 70% on average. As could be anticipated,
perfect identity was mostly found in the cross-linking
domains and in exons readily known to be highly con-
served among species, i.e., exons 33 and 36 [64]. In
contrast, in exons encoding for hydrophobic domains,
perfect identity was much more localized. This type of
'discrete' identity is well illustrated for exons 5, 20 and 30
(figure 1) where short domains of identity could be evi-
denced. Interestingly, in most cases, the conserved amino
acid sequences were: either G or P alone, or GX or XG
(where X = G, A, V, L or I), or PG, or a combination of
those. The most conserved stretches were GG and PG
suggesting that peptide sequences belonging to the family
studied by Urry and Tamburro should have an important
role.
2.2. Secondary structures predictions
The GOR III method [65] has been applied to the six
tropoelastin sequences. The predicted secondary structure
contents are compiled in table I and the attribution of
Elastin structures and their function 983

. . . . . . . .

HT~: V l P I G AI~ P G G - F YIPIIG A G I L I G I A L G I AlL GIPIGIGIK P I L I K P~V P

RT~: VlPl~ ~iv p ~ G . GL~G~VP VlPIP ~ GIIIGI~ G L G - AlL GIPIGIGIK PIPI K PIP A
MTE:
STE:
CTE: I!"i V P
V L
V PIG A l l
-
V
- - -
P G G
PGG liiil G L P GG V P G

:
v Y YIPUG A G I , p i G
F FIP G A G I L I G I G
F FIP G A G I V [ G I G
G G
L
L
G
V
A
-
-
AlL
GIL
L G A GIL
GIPIGIGIK PIPI K
GIPIGIVIK PI^I K
G[ALGJV K L K ~ L ~ K
P~G
PI G
PIG
A
V
V S G

RTE: . . . . GA F GA P G G - P AG L S Y - -AS - V L V PGGGA G Y A

MTE: - - - G T F G AIGIPIGIGIL GIGIA I - p A G L G A F F A G G A L V P G G A A G Y A
STE: VIGIPIGI-IL GI-IA,E S L L P AGA F - F F GAGGGAAG Y A
CTE: G~I. IG P L G L Q P G AI_G.[L L.GJ
. G
A LL.LJ.__~ V - -L.G.J L I G I A F P G A A F . . . . . . . . A A LIK A AIAIK AG I

HTE: V . . . . . . . . . P G V . . . . . . . . . . . . . . . . . . . . . . . . . . . . W S A A
RTE: . . . . . . . . GIGGVPGGVGVGGVPGAVGVGGVPGAVGGIGGI V S T A
MTE: VGGVPGGVGVGGVPGGVGVGGVPGGVGVGGVPGGVGGIGGI ST A
L O,OVGG . . . . . . ,GGV . . . . . . . . . . . . . . . . . . . . . . . . . . .

CTE: V G G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I IG G L GIG V G V G V P

. . . . . . . v . . . . o v
RTE: . . . . . . p L G A V G A G G G V L V V - T G AIIR F P G I V I G V L P G V P T
MT~ . . . . . . . p v G A ~ G A G G I ~ PIGI K V P I I G I V l G I L I P GIV V - T G AgR F P G I V l G V L P G V P T
STE. . . . . . . G A G V G V I ~ PIGIK V P G I V l G I L I P GIV Y - T G AIR F P G I =1 G V L P G V P r
CTE: G G L G V P " " G A A G KL.K._P.JP I E V P G I A I G I I [ P GIA F G AG I[RFPGIVIGVLPGVPT

BTE: , ~ v~ P ~ , ~ 1 ~ G~ ~ m ~ I ~- I- I1G1 v2 G P F G G O O P G V P L G ~ P K ~ p K L P I I ~ I - 13
~IGI-~YKTGKLP i l~F
lifE: A V P A V A I F I A I G I PII G V G P F G G P Q P G V P L G Y P K A P K L PnGIG V I G I L P l Y r r G K L P
Y
RTE: v v A v G AIFISlG ' PIpVGPFGGOQPGVPLGYP KAPKLPIIGIGVIGILPIYTNGKLP
-
MTE: T V A A G A I F I S I G I P~G V G P F G G a Q P G V p L G y p K A p K L PUGIG YIGIL p l y r N G K L p
STE: A V P A G R I F I A I G I PIG V G P F G G a Q P G V P L G Y P K A P K L P GIG Y I G I L PI Y S T G K L P Y I G I Y
CTE: TIGIIIKIAIKIGIP G I A I G GI A I F I A I G I PI . . . . . . . . . . . . . . . . . . . . . . . . GIG Y J R L . ~ . J F . . . . . V NGL~ L

BTE:
HTE: G P E G V A A G K A G Y P T G T G V GIPIQ A AIAIAIA A A A AA V - - GV
RTE: . . . . V A G G K A G Y P T G T G V GISIQ A AIVIAIA A - - A GGG V - G
MTE: v AGGKAGYPTG VGISlQAAIAIAIAA GA V - - G
STE: G P o G V ,, G K,OVP O V O ;1: : :I:N °" v .%;°
CTE: G P G G I L K A G Y P T G V G AIQ A AIALA j . . . . . .

8TE: A P[-G1A I["~ I[-G'IG . . . . . . . . . . . . . . . . . . . . . . . .

HTE: V P ~ A lip GI,IGIG AIIGIVlGIT - AAIAAAIAIAAA K A A - V . . . . . . . . . . . . . . . . . . . . . . . .

RTE: G A A lIP GIIIGIG TIIGIAIGIT - AAIAAAIKIAAAKAA- Y . . . . . . . . . . . . . . . . . . . . . . . .
MTE: G A A lip GIJIGIG
STE: A P A liP GI,IGIG A I l'lA l A A A A A A A K A A -. vY . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . .

CTE: V G V VIP GIVIGIV P L.G_JAI GI V - -IAAAIAIAAAKAAA AGAYGAGVLPGAGGVPGVVPGVGVV

BTE: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
HTE: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
RTE: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MTE: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
STE: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CTE: P G L V P G VGG I PGVAGVGTPAGAAAAAAKAAKYGAGVPGVGVPGVGIGGVPGVPGVPGVP

Figure 1. Alignment of tropoelastins sequences. The signal peptide sequence has been removed. The alignment was performed using
the identity matrix and exon boundary constraints were applied when possible. Double vertical bars indicate exons borders. Exon
number as defined by Rosenbloom and colleagues [53] is reported. The exons boundaries for STE and CTE are not labe]ed as they
have not been established yet. - , gap. Boxes indicate perfect amino acid conservation among the six species.
 GO

.m .... m ~
~ .... ~ . . ~

~ , , , , , , <: ¢3 ¢3 C) . G) , , , , ,
< , , < <
~ , , , , , , , , , ,
, , ~ , , , G) , ~ G')G~ ,
G3, , , G3, "D , , , , ,
), ). ), > < , , ~ , , ,
0 ~ , , , , , G) . , . , ,
r- , , , -- ,
< , , , , , , , , , ~ , ¢C , t , , ,
< ~ , > < < > ) . , C) ~. ~.
m ~ , , , , , • •.g , , , , ,
"o , , , -g ,
; ~ 0 , 0 0 0
, , , Q , ~ , , , , ,
.c=. < < - - , , < ,
<, , , < , ~ , , , , ,
~0
Q, > ) , > , < , , , , , '13 , , , , ,
), , , , , , < <
C}
G3, C) G) G') , G) , , , , ,
"11 ~ 0 0 0 > '
r- , , , , , ¢C , , , , ,
C~ < , , , , ,
< , , < "xl . , , , ,
), &3 , , , , ,

C} , , . , ,
, , , , ,
< , , , , , ~3 , , (n
> , < , , , <
< r" r" < G3G3 ¢3 G3 ¢3 , , , , ,
C}
G) ~ ) C} G) G) , , , , ,
<
"o'Io "g "g "g , , , , ,
..g > . . . . , < G3 , ,

r- , , <: "0 , , , . ,
< , , , , ,
), , , , , , G} , , , , .
-~ , , , , ,
f- "n "n ~ r'-
~G3 C) C ) ~
, G3 C~ G') G"}
< < - - < < •.g , , , , ,
C) G3
, > ~ > > >
"g "o " g "10 " g G) , , ,~, ,
G3 C) G3 C) G) , > ~ > > >
< , . . . .

•.g , , , , ,
C} G')
, &') ~ C} G)
r- < C}C) G3 G) C) G) , . . . .
, < : < < < O X X X X X < < ~ < < < <
, , , , , ¢C , . , . .
, " g " g "Io " o , if) ~ ffl ff~ if)
, . ---- . , < . , , , .
, G) c ) c ) G3 , <ff~ff) < <
G) , , , ~ , •i o , , , , ,
, .-n - n - n "11 > > > ~ > > , _>_< <_> >
< , , , < , C} C) G) C} < C) C} , , , , .
G) G3 ~ C) C)
L. . . . . . 1 ~ , , , , , <: . , , , ,
G3 , , , ~ ,
)=, , , , -- , G) . . . . ,
J~ < . , , , ,
, , , , ).
~ G) ¢ ) G3 " o , G) , , , , ,
, , fflff~ , , - - < < < - -
, , , , o
I. . . . . . I ~ , , , , , C) , . . . .
, , . , rn
G) G3 C) C~ "I0 , , , , ,

, , , , < < < < < <

, , , , ,
, , , ,
, , , , , < < , , < )= . , , . ,
, , , ,
"-!'<I 0 , 0 0 0

G) G) , , G3 ~, . . , . .
, , , . r- < < , , < ¢ C

"~ G') G3 C} G) ~
:~ , . , , ,
. . . . "o < < < < <
). , , . , ,
, , , , m , ~' &3 G) ~ , <
) . . . , , ,
, , , . r- "g "g < "0 "0 "0
G) G') G) G) G3 7. , , , , .

, , , , ill ~ < < - - < << , , <<

G') C) ¢ ) G) G) C~ , , , , ,
, , , , c)
L* ~ ° ~ ~ ~J X ~ K X ~ , -- G) G)-- < 7, , , , . ,
. , , , o
I. . . . . . I ~. , . . , ,
. . . . •13
C) C~ C) C) "11 . , , , .
, , , ,
X X X X X
, , , , (n < ~ < < <
-- -< . < ..< - - G') C} G3 G) ~ G)
, , , , (n ~ < < - - <
<; < G3 G) < <
, , , , o < > > < < {:)."

0 0 0 0 ~ I. . . . . . I I. . . . . . I
C) , G') C= , C~
, , , , r. < < < < <
G3 G3 ~ ) G) G3 , , ---- . ,
, , , , ..g
< ~ < < < ,
, , , , (n . , > > £ ~
C) C) G) C} C) G) , C) C) , 0
< , G3G3 , >
. . . . "0 I. . . . . . I
Elastin structures and their function 985

BTE. . . . . VPGTLAAAKAAKF~ . . . . . . . . . . . PGGV-GALGGVGDLGGA-GIPGGVA~

~E:SSP VPGALAAAKAAK YG . . . . . . . . . . . . . . VPGVLGGLGALGGV GIPGGVVG
RTE: AVPGSLAASKAAK YG AAGGLGGPGGLGGPGGL GGPGGFGGPGGLGGVPGGVAG
~E: AVPGSLAASKAAK AAGGLGGPGGLGGPGGL GGPGGLGG . . . . AGVPGRVA
STE: . . . . . . VPGTLAAAKAAKFA . . . . . . . . . . . . . PGGV-GALGGVGDLGGA-GIPGVGGG
CTE: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

BTE: V G P A - - AAAA - AKAAAKAAQF~ ~ L - G - - ~! V . . . . . . . ~ V~ A . . . . . . . . . . . . .

HTE: A G P A - - AAA A AA KAAA K AAQ F~L VGA A L . . . . . . . . . . . . . . . . . . . . .
RTE:GAPA--AAAA-AKAAAKAAQ y L G . . . . . . . . A AGGLGAGGLGAGGL
MTE:AAPPAAAAAA-AKAAAKAAQ y L G . . . . . . -IGGLGIAIGGLGIA'GGLGAGGLGAGGL
STE:VGPA--AAALGAKAAAKAAQ G . . . . . . . -P-IIG
GGGLLG
GIIVVIIG
GGGLLG
G]I A : : : : : : : : : : : : :
CTE: . . . . . ; . . . . . . . . . . . . . . VPGA~VPGVGGI

. . . . . . . . . P A V L - VSPAAAAKAAKFGAAGLGGVL-GAGQPFPIG VAAR
HTE: . . . . . . . . . V P V G L IPPAAAAKAAKY[IGAAGLGGVLGGAGQ-FPLGI~VAA
RTE: G A G G L G A G G V I P A V L - VSPAAAAKAAKYIIGAAGLGGVL.GA.RPFPGGI~VA A
MTE: G A G G L GAG G L G A G L A !~!VSPAAAAKAAKYIIGAAGLGGVL-GA-RPFPGGI~VAA
STE: - V G G L G A - - V P V L VSPAAAAKAAKFGAAGLGGVL-GAGRPFPIGGVAA
CTE: . . . . . . V G G L - G L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AGVG

B T E : ~ L ~ F GG-AGG*LGV
HTE: L F . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIGKIAICGRKR
RTE:
~E: ~LIS
L Y
P IIY~G
GGGAGG-LGV
G G A G G - L G V GGKPPKPYGGALGALGY
OlIOKP.KPYoo,AoY-
-FIGKISlCGRKR
FIGKISICGRKR
STE:~LISPIIF~GG-AGG-LGV GGKPPKPFGGALGALGF LIGKISlCGRKR
CTE:~VISPIIF~GG-VGGQLGF GGKPPKTYGGALGALGFRGL~VG~AQGI_9_~YICGRKR

Figure 1. Continued.

BTE substructures is provided (figure 2) as an example. plot) exhibited a difference often lower than 50 cnats
Ten helical domains (20%) corresponding to the AK suggesting that both were possible. This finding brought
cross-linking domains alternated with the 'elastic' do- additional evidence that elastin [3-structures were certainly
mains of the molecules which appeared to consist of short short and/or distorted ones and that an equilibrium be-
and/or irregular [3-strands (30%) mixed with undefined tween these conformations and others could exist [54, 55].
structures (50%). These results were in good agreement
with those reached formerly using other predictive meth- 2.3. CD and FTIR spectroscopies
ods [54], but the new and more accurate quantitative The secondary structure of BTE was studied in the
estimations exhibited less ordered structures (helix + solid state by FTIR and in aqueous solution using
[3-strands). CD [55]. The quantitative results underlined that a sub-
The analysis of the directional information curves for stantial amount of extended structures was present in both
each of the three substructures (figure 3) underlined that states: 5% a-helices, 50% [3-sheets and 45% undefined
the helical segments were easily identified as peaks of conformations. Importantly, the analysis of the FT1R
high positive information (figure 3, upper plot). In con- spectrum allowed to demonstrate that antiparallel [3-sheets
trast, in the hydrophobic domains, the information in favor were present. The CD spectrum of BTE was very similar
of [3-strands and unordered conformations (figure 3, lower to that of bovine a-chymotrypsin and porcine pancreatic
elastase [66] suggesting that tropoelasfin could possess a
structure similar to theirs. According to their crystal
Table I. Secondary structure percentage contents of the tropo- structures [67], bovine c~-chymotrypsin and porcine pan-
elastins according to the GOR III method. creatic elastase possess short and/or irregular antiparallel
a 3 v p-sheets instead of regular ones. Thus, such structures
should also be present in the conformation of tropoelasfin
BTE 22 30 48 and, given their irregular nature, they could be involved in
STE 22 31 47 structural transconformations as suggested by the predic-
HTE 24 29 47 tions.
RTE 19 37 44 Although the overall agreement between theory and
MTE 17 38 45
experiment was rather good, several discrepancies are to
CTE 16 32 52
be discussed. First, the extended structures of tropoelastin
986 Debelle and Alix

i0 20 30 40 50 60 70 80 90 i00

BTE I GGV~GAV~GG~PGG~FFPGAGLGGLG~GGLGPG~KPAKPG~GGL~GPGLGAGLGAL~GAFPGALVPGGPAGAAAAYKAAAKAGAAGLGVGGIGGVGGLGV
GOR III ..... / ........ //// . . . . . . /// ............... /// ........ // ....... // ..... 0000000000000000///////////////

BTE i01 STGAw~QLGAGVGAG~K~GKV~G~GL~GVYPGGVL~GAGARF~GIGVLPGV~TGAG~KPKA~GGGGAFAGIPGVGPFGGQQ~GVPLGY~IKAP~LPAGY

GOR Ill //-////// ..... // .................. /// .... /---//// ........ / ......... ////-/// .........................
t

BTE 201 GLPYKTGKLPYGFGPGGVAGSAGKAGYPTGTGvGPQAAAAAAKAAAKLGAGGAGVLPGVGVGGAGIPGAPGAIPGIGGIAGVGAPDAAAAAAAAAKAAKF

GOR III .... /--/--/ ...... ////////--//--/--0000000000000000---//////// ............. ////////--0000000000000000

BTE 301 GAAGGFPGVGVPGVGvPGVGVPGvGvPGVGvPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGAVSPAAAAKAAAKAAKFGARGGVGIGGIPTFGVGPGG

GOR III OO ...... ,,-,--,---,,---,,---,,---,,---,,---,,---,,---,,---,, .... 0-000000000000000/--//////////// ....

BTE 401 FPGIGDAAAAQAAAAAKAAKIGAGG•GALGGLVPGAPGAIPGVPGVGGvPG•GIPAAAAAKAAAKAAQFGLGPGVGvAPGVGVvPGVGVVPGVGVAPGIG

GOR III .... 0000000000000000000--/////// ............ / ...... / - - O O O O O O O O O O O O O O O . . . . . . ///--////--////--////---/

BTE 501 LGPGGVIGAGVPAAAKSAAKAAAKAQFRAAAGLPAGVPGLGVGVGVPGLG•GvGvPGLGVGAGVPGFGAVPGTLAAAKAAKFGPGGVGALGGVGDLGGAG

GOR III ..... ////--OOOOOOOOOOOOOOOOOOOO . . . . . . . . ///////--///////--////---/--/--000000000000 .... ////////// ....

BTE 601 IPGG•AGVGPAAA•AAKAAAKAAQFGLGGVGG•GVGGLGAvPGAVGLGGVSPAAAAKAAKFGAAG•GGV•GAGQPFPIGGVAARPG•GLSPI•PGGAGG•

GOR III -/--///--0000000000000000//-////////-///---//// .... O O O O O O O O O O O O O O O / / / / . . . . . . . /--/// ....... /// .......

BTE 701 GVGGKPPKPFGGALGALGFPGGACLGKSCGRKRK

GOR III II .......... II1-1 ...... I ..........

Figure 2. GOR III pure prediction of BTE secondary structures. The first row corresponds to the sequence of BTE (734 residues). The
second one, GOR III, corresponds to the predicted structure. The codes are: O, a-helix;/, fJ-strand; -, undefined conformation.

had been underestimated in the predictions. In the crys- sequently it may be statistically overestimated. Addition-
tallographic databases from which the prediction methods ally, the confidence range of structural estimations from
are derived, irregular structures as those encountered in spectroscopic data is 5%. However, these two points are
tropoelastin are not frequent and therefore their prediction not sufficient to explain such disagreements between
is somewhat biased resulting in a percentage content 20% predictions and experimental results• So, the a-helical
lower than the experimentally determined one. Second, cross-linking domains of the tropoelastin could be smaller
the predicted helical content was much more important than those predicted. Interestingly, this would mean that in
than that determined experimentally, 2-4 times larger. The the isolated tropoelastin molecule, the cross-linking do-
a-helical conformation is well known to be the predomi- mains, which are conventionally described as stable
nant substructure in the crystallographic databases, con- (x-helical structures could also experience some deforma-
tion due to the internal dynamics of the molecule and
therefore be shorter than previously thought•
90O 2.4. Molecular model of tropoelastin

A A A AAn A^AA I Following the use of the LINK method [68-70], the
global secondary structure of BTE determined by CD [55]
was adjusted using a GOR III prediction. This way, the
1 101 201 301 401 501 601 701
distribution of the secondary structures corresponding to
the experimental data was addressed (data not shown).
90O
The distribution of the structures was very similar to that
provided by. the pure predictions but the helical structures
occurring in the cross-linking regions were shorter. Inter-
estingly the extended structures found in the 'elastic'
-300
domains of BTE appeared more irregular after adjustment•
~0o
1 101 201 301 401 501 601 701 In fact, the fitted GOR III prediction provided a secondary
sequenceof BTE structure distribution for BTE which was very close to that
exhibited by both bovine ct-chymotrypsin and porcine
pancreatic elastase using the same secondary structure
Figure 3. Directional information plots of the GOR III predic-
tion of BTE secondary structures. Upper diagram, directional definition [65], and consequently a fJ-class folding type
information of the helical conformation. Lower diagram, direc- was proposed for BTE [55]. We would like to point out
tional information for the extended and unordered conforma- here that the structures found in the 'elastic' domains of
tions. the tropoelastin are continuously interchanging•
Elastin structures and their function
Importantly, the model proposed for tropoelastin [55]
can be reasonably transferred to all of its isoforms. Indeed,
given the clear separation of tropoelastin functional do-
mains and their cassette-like encoding, alternative splicing
would certainly exert few (if any) influence over the final
structure of the molecule. For example, the suppression of
a 'hydrophobic' exon would merely result in a shortening
of the corresponding hydrophobic region, but would not
modify its substructures because the repeating nature of
the sequences encountered would be preserved. Likewise,
the scarce prolyl hydroxylation occurring in tropoelastin
could hardly have any impact over the global structure of
the molecule. Our feeling is that these subtle modifications
could be relevant only at the level of local structural
modifications rather than large ones that could affect the
structure of the monomer and its elasticity. The nature and
influence of these local events could possibly be important
in elastin-other macromolecules interactions. However if
this holds for 'hydrophobic' exons, we must underline that
splicing patterns of cross-linking ones could have a
profound effect on the final architecture of the insoluble
elastin molecule.
3. The structure of elastin
Elastin is constituted of cross-linked tropoelastin mol-
ecules and the details of events leading from isolated
monomers to the insoluble polymer have been described
elsewhere [71]. However, we would like to underline
several points. First, the fact that an effective chaperone
prevents tropoelastin premature intracellular aggrega-
tion [72] strongly suggests that, somehow, the nature of
tropoelastin structures are important to elastin function.
Second, as the microfibrillar component plays a critical
role in the correct alignment of the monomers [73], a
regular arrangement of the tropoelastin molecules could
be expected in elastin. Finally, although the first formed
ones have been recently identified [74], the distribution of
the cross-links remains unknown. They are probably
mostly intermolecular but the possibility of intramolecular
cross-links can not be excluded.
3.1. NIR FTR and FTIR spectroscopies
The analysis of the near infrared Fourier transform
Raman (NIR FTR) spectrum of bovine elastin (BE)
(figure 4) revealed that helical structures were present in
the conformation of the molecule because the band at
934 cm -I is typical of helices [75]. Additionally, a sub-
stantial amount of [5-structures was evidenced because the
amide III band maximum (1248 cm -I) occurred in the
frequency domain of these structures [76]. The quantita-
tive estimates derived from the decomposition of the
amide I band (1661 cm -~) confirmed these points and
were consistent with those obtained for human (HE) and
987
Table II. Secondary structure percentage contents of elastins in
the solid state as derived from the analysis of the amide I band
of their respective NIR FI'-Raman spectra.
a ~ u
BE" 9 43 48
HE b 8 36 56
RE 12 33 55
aAdapted from [56].
bAdapted from [77].
rat elastin (RE) (table II). Thus, the structure of elastin in
the solid state appeared to consist of about 10% a-helices,
40% [5-sheets and 50% undefined conformations.
These values were very close to those obtained for-
merly for BTE: 5% a-helices, 50% p-sheets and 45%
undefined conformations [55]. This finding strongly sug-
gested that upon polymerization, the global structure of
BTE did not change significantly and that the fibrous
elastin polymer would be constituted of cross-linked
globular tropoelastin monomers. Interestingly, the slight
increase of the helical content could be due to the mutual
stabilization of the cross-linked helical domains. Like-
wise, the higher content of unordered structures in elastin
conformation suggested that the polymer could possess a
higher entropy than its monomer.
The analysis of BE [56] and BTE [55] FTIR spectra
also demonstrated that the structures of the two proteins
were very similar. Their conformation sensitive bands
were found at very close positions: amide I at 1659 cm -1
and 1658 cm -l, amide II at 1538 cm -1 and 1540 cm -1, for
BE and BTE respectively. However, the distributions of
their conformational states varied as indicated by the
positions of their amide A bands (3322 cm -1 for BE,
3299 cm -1 for BTE), the frequency of which is directly
correlated with the mean H-bond length in which the
peptide NH are involved [78]. The higher the frequency,
the looser the H-bond. These findings strongly suggested
that BE exhibited a higher entropy than BTE.
3.2. Structural model of elastin
The fact that BE appeared to possess a higher entropy
than its monomer raised the question as to whether or not
the model proposed for the free tropoelastin molecule
could be relevant for the polymeric protein. The use of
bovine n-elastin (BKE) NIR FTR global secondary struc-
ture contents (13% a-helices, 46% B-sheets, 41% unde-
fined) to fit a GOR III prediction (LINK method), allowed
us to obtain the secondary structures distribution of
polymerized BTE. The structural contents of BKE were
preferred to those of BE because elastin is insoluble
whereas tropelastin is soluble. As the sequence used for
the fit was that of BTE, we thought that the use of data
988 Debelle and Alix

A
"1"

"r"
v

a)

i
I
, .-.

I (,/1 ~, ~ ~i ~ I--- ~ I~
2, ~. er ,o. ~ .~ , -
"- "~" ~ ~ A~ -~ "- ~..~., ^ '~
" ~ I1~ "-~ ~ HI- "~ /~ ~1
(~1 j= t/1 i j 0~ I

0 ~ r'l I

w
5do 1o;o 15bo
Wavenumber (cm q)

Figure 4. NIR FT Raman spectrum of BE in powder in the frequency range 500-1750 cm -1 and assignments of the observed spectral
features, v, stretching; 6, deformation; G-G-T, gauche-gauche-trans; (~t), band at 934 cm 1 corresponding to the C~,-C stretching mode
of ordered a-helices [75]; (~), amide HI band maximum at 1248 cm -~ in the frequency domain of [3-structures [76] (from [56]).

pertaining to a soluble, cross-linked polymer, i.e., BKE,
could be an acceptable compromise between the two
natural forms of the protein.
The distribution of the structural elements along the
sequence of BTE when it is polymerized to form BE
(figure 5) was consistent with that obtained for the isolated
monomer (data not shown). This finding strongly suggests
that the structure of the isolated tropoelastin was most
certainly preserved in the elastic network. Indeed, as noted
above, the influence of cross-link formation resulted in
few structural modifications. However, very importantly,
their formation seems to strongly affect the dynamics of
the molecule. For instance, the helical cross-linked do-
mains appeared to stabilize each other resulting in a
greater local rigidity whereas a notable increase of entropy
could be observed in the hydrophobic domains as the rate
at which their substructures interchanged was raised.
According to these results, a simplified description of
the tridimensional structure of elastin was proposed (fig-
ure 6). In this schematic model, elastin was constituted of
a tridimensional network of globular, regularly aligned
tropoelastin molecules cross-linked with their neighbors
and possibly themselves. This type of spatial organization
has been confirmed using atomic force microscopy for
bovine ct-elastin [79] and recombinant HTE [58] coacer-
vates. Within the assembly, the structure of each tropoelas-
tin molecule consists of: i) rather rigid c~-helical segments
where the cross-links are formed; ii) large hydrophobic
domains which are responsible for the elastic properties of
the polymer and whose conformation consists of
fS-structures and undefined conformations rapidly and
continuousl3, interchanging; and iii) a substantial amount
of hydration water molecules [16, 56] which are tightly
bound [16] to the polypeptide chains. When elastin swells
in water, bulk solvent water molecules (B) diffuse in the
interstitial spaces as indicated in figure 6.
We emphasize here that the schematic description
provided infigure 6 is intended as an example of what the
general nature of elastin structure might be at a given time.
Indeed, due to the constant internal dynamics of its chains,
elastin structure is always changing and the static descrip-
tion provided by figure 6 could be misleading.
Elastin structures and their function 989

I0 20 30 40 50 60 70 80 90 i00
BTE 1 GGVPGAVPGGV~GGVFF~GAGLGGLGVGGLG~GVKPAKPGVGGLVG~GLGAGLGALPGAFPGALVPGGPAGAAAAYKAAAKAGAAGLGVGGIGGVGGLGV
LINK .... // ........ //// ..... ///// ............. //// .... //--// ....... /// ....... 000000000/-/////////////////

BTE i01 STGAVVPQLGAGVGAGVK•GKVPGVGLPGVYPG•VLPGAGARFPGIGVLPGVPTGAGVK•KAPGGGGAFAGI•GVG•FGGQQPGVPLGYPIKAPKLPAGY

LINK ///////// ..... // ....... /// ........ /// .... /---//// ........ / ......... //////// .........................

BTE 201 GL•YKTGKL•YGFG•GGVAGSAGKAGYPTGTGVG•QAAAAAAKAAAKLGAGGAGVL•GVGVGGAGIPGA•GAIPGIGGIAGVGA•DAAAAAAAAAKAAKF

LINK ---II--IIIII ..... IIIIIIII--II-III--II00000000011 ..... IIIIIIIII--I ....... I-IIIIIIII .... 00000000000011

BTE 301 GAAGGFPG•G•PG•G•PG•G•PG•GVPGVG•PG•GVPG•G•PG•G•PG•GV•GVG••GVG•PGAvSPAAAAKAAAKAAKFGARGG•GIGGI•TFGVGPGG

LINK II ...... IIII-IIII-IIII-IIII-IIII-IIII-IIII-IIII-IIII-IIII-II .... I--00000000000111111111111111111 ....

BTE 401 F~G~DAAAAQAAAAAKAAKIGAGGVGALGGLV~GA~GAI~GV~GVGGVPGVGIPAAAAAKAAAKAAQFGLG~GVG~APGVGVVPG~GwPG~G~APGIG

LINK --I---00000000000011111-IIIIIIIII ........ I-II ...... II-I000000000001111 .... IIII--IIIIIIIIIIIIIIII--II

BTE 501 LG•GG•IGAGVPAAAKSAAKAAAKAQFRAAAGLPAGV•GLGVGVGV•GLGVGVGVPGLGVGAGVPGFGA•PGTLAAAKAAK•GPGGVGALGGVGDLGGAG

LINK ..... IIIII .... 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 ......... IIIIIIIII-IIIIIIIIIIIIIIIII-III .... I0001111 ..... IIIIIIIIII--I-

BTE 601 IPGGVAGVGPAAAAAAKAAAKAAQFGLGGVGGLG•GGLGA••GAVGLGGVSPAAAAKAAKFGAAGLGG•LGAGQPFPIGG•AARPGFGLSPIFPGGAGGL

LINK -/--/// .... 00000000000///////////////////-///// ...... 000000//-/--////// ...... /-//// .... /--/// ...... /

BTE 701 GVGGKPPKPFGGALGALGFPGGACLGKSCGRKRK

LINK // .......... ///// ..... /// .........

Figure 5. Distribution of BTE secondary structures after adjustment of the GOR III prediction using the global secondary structure
contents determined experimentally for BKE in the solid state. The first row corresponds to the sequence of BTE (734 residues). The
second one, LINK, corresponds to the adjusted GOR III prediction. The codes are the same as those defined in the legend tofigure 2.

4. Structure-elasticity relationships assessed by the examination of the position of the water

4.1. Water swollen elastin
Elastin is elastic only when it is swollen in water [60,
61, 80, 81] and the fact that the reason for this necessity is
still not understood indicates that water should deserve
particular attention.
The action of solutes on the structure of elastin is
indirect and mediated through its hydration shell [82].
Thus if one regards bulk solvent water molecules as a
particular solute, their plasticizing effect [62, 63] should
also be processed through the hydration shell of the
molecule. Consequently, the system giving rise to elastic-
ity, i.e., water swollen elastin, would be constituted of
three 'phases': i) hydrophobic and cross-linked polypep-
tide chains; ii) hydration water, which is tightly bound to
the polypeptide chains [16]; iii) bulk solvent water, which
swells the polymer in vivo. In keeping with this 'triphasic'
description of the polymer, elastin structure-elasticity
relationships could be explained as follows.
4.2. Influence of hydration water on the structures of
elastin
In the light of experimental and theoretical results,
water mediated structural equilibria have been proposed
for the conformations found in the 'elastic' domains of
elastin, say extended, ~-turn and random coil substruc-
tures [40, 41, 43, 54, 55]. The molecular role of water in
these phenomena is not straightforward but is possibly
molecules in crystals.
In the crystal structure of the elastin-like synthetic
peptide Boc-VPGVG-OH [36], the occurrence of hydro-
gen bonds with hydration water seems to disrupt the
pleated structure of the peptide. Likewise, a recent inves-
tigation of the location of hydration water molecules in
protein crystal structures [83] demonstrated that the water
molecules could disrupt type II [5-turn geometries by
insertion between residue 1 and 4 thus preventing the
formation of the usual 4 ~ 1 hydrogen bond. Such possi-
bilities also exist in the case of elastin [42, 45]. So, the
occurrence of a hydration water molecule seems to be
unfavorable to the formation of hydrogen-bonded, regular
structures.
This point is extremely important in the case of elastin
as it means that the water molecules of its hydration shell
would raise the conformational entropy of the molecule.
But, at the same time, and it is a paradox, as those
molecules are tightly bound to the polypeptide chains,
they would also certainly constrain them by limiting their
mobility. In short, hydration water contributes to the
conformational entropy of the elastomer but does not
permit to reach a mobility level allowing elasticity. This
explains why dry elastin is brittle.
4.3. Importance of bulk solvent water
When bulk solvent water swells elastin in vivo, water
molecules diffuse through the whole network and fill the
interstitial spaces between the globular tropoelastin mole-
990 Debelle and Alix

Wllllllllll_ ]flI~PIIPPI_w g¢
w w ...........

v w
H2N-.,w,ZZ.J wW/

~w VVV--~w~ COOH
qs-srJ

Figure 6. Simplified molecular model of hydrated elastin. Lower pan. A small part of the tridimensional elastin network is presented.
The globular tropoelastin molecules (circles) are arranged regularly. They are covalently bound to each other by intermolecular
cross-links (crosses). Bulk solvent water molecules (B) occur in the interstitial space. Upper part. Each tropoelastin molecule of the
network (gray circle) possesses an ~ class structural folding. The a-helical cross-linking domains are at the surface of the molecule
to permit polymerization. The hydrophobic regions consists of an alternance of ordered and unordered conformations. The hydration
water molecules (W) are found at the surface and within the folded monomers. Together with bulk solvent molecules, they contribute
to the continuous and rapid internal chains dynamics of elastin ( s e e f i g u r e 7). Thus, as a fixed and static image could be misleading,
we underline here that the provided description should be regarded as an example of what the general nature of elastin could be at
a given time.

cules bringing bulk solvent water molecules in contact
with the polypeptide chains and their strong hydration
shell.
The consequence of this is two-fold. First, the polypep-
tide chains have their hydration shell fully saturated by the
transfer of some molecules from the solvent to the
hydration water 'phase', enhancing the conformational
entropy of the system. Second, as is usual for neighboring
water molecules, hydrogen bonds are formed between
bulk solvent and hydration molecules, and consequently,
Elastin structures and their function
the hydrogen bonds between the hydration water mol-
ecules and the polypeptide chains become looser allowing
higher mobility.
In this proposal, solvent water acts as a plasticizer for
elastin, by increasing the mobility of its chains as it was
suggested [62], and that effect is processed through its
hydration shell as it should be [82].
4.4. Dynamic structures and elasticity
Two types of structures can be found in the hydropho-
bic regions of elastin: i) ordered conformations that are
stabilized by internal hydrogen bonds like the [5-strands
and the [3-turns; and ii) unordered conformations also
termed random structures. Additionally, they are thought
to be involved in structural equilibria in which hydration
water would play a critical role [40, 41, 43, 54, 55]. We
emphasize here that the ordered structures of elastin are
extremely labile when compared to those encountered in
other proteins. Consequently the usual picture of fixed,
blocked structures should be precluded here. Keeping this
in mind, elastin structure-elasticity relationships could be
summarized as presented in figure 7.
In the relaxed state (figure 7, upper part), the structural
equilibria are in favor of the unordered conformations.
Bulk solvent water (B) plays an important role (upper-
case) by interacting strongly with the hydration water (w)
of the molecule and therefore it prevents it from restrain-
ing the mobility of the polypeptidic chains (lower case).
Water swollen elastin is at considerably high entropy and
its chains experience a very high mobility [22].
When the swollen polymer is stretched (figure 7, lower
part), the constitutive tropoelastin molecules unfold [21].
Likewise, bulk solvent water is mostly excluded from the
interstitial space and consequently its interaction with the
hydration shell of the molecule is greatly reduced (b, in
lower case). The polypeptides chains are constrained
along a predominent direction (that of the stretching force)
and they come into contact with each other. In this
condition, the formation of regular structures, as for
example hydrogen bonded [3-sheets is made possible.
Additionally, with the exclusion of bulk water, hydration
water is given the possibility to greatly restrain the
polypeptidic chains (W, uppercase). Both phenomena
contribute to pulling the structural equilibria towards
ordered and restrained structures. Following this molecu-
lar rearrangements, the entropy of the system drops
considerably.
When the stretching force is removed, elastic recoil
occurs following a classical mechanism of elasticity [3] as
the tropoelastin molecules fold and the elastic network
swells anew.
5. Conclusion
The structures of elastin have been re-evaluated and
reviewed and consequently, a molecular model has been
991
B
Relaxed
ordered w unordered i
restrained "~"-~ I mobile
Stretched
ordered W unordered
restrained ~ mobile
Figure 7. Water mediated structural equilibria proposed for the
hydrophobic domains of water swollen elastin. When the elastic
network is relaxed, bulk solvent molecules interact strongly (B,
uppercase) with the hydration shell water molecules (W) for-
ming hydrogen bonds (heavy dotted line). This prevents those to
restrain the polypeptide chains (w, lowercase) and consequently,
mobility is enhanced. Entropy of the system is very high and the
structural transitions occuring in the hydrophobic domains (dou-
ble inverse arrows) favor unordered structures (strong arrow to
the right, weak one to the left). Upon stretching, the tropoelastin
molecules unfold and regular hydrogen bonded structures are
favored as their chains orient following the axis of the stretching
force. At the same time, the bulk solvent 'phase' is excluded
from the interstitial space of the network and its interaction with
the hydration shell is considerably reduced (b, lowercase; weak
dotted line). Hydration water can exert a greater influence (W,
uppercase) and restrain the polypeptide chains (strong arrow to
the left, weak one to the right). Following these molecular
events, the entropy of the system drops dramatically and a
classical entropic mechanism [3] of elasticity is allowed.
proposed for the water swollen elastomer. Additionally,
the structure-elasticity relationships of the polymer have
been tentatively explained by molecular equilibria. As
they are based on a compilation of experimental and/or
theoretical evidences in favor of it, we feel that our
proposals could provide an accurate description of the
hydrated structures of elastin and of their dynamics.
Additionally, we are greatly supported in this feeling
because our models are fully consistent with the merging
new theory of elastin elasticity [84]. However, we should
underline that the fractal aspects of the polymer [85, 86]
were not taken into account here.
On the basis of these models, pathological states
leading to modifications of elastin elasticity and those
promoted by the binding of ions or small molecules, like
Ca 2÷ or fatty acids, can be envisaged differently and,
hopefully, be described at the molecular level [87].
992
Acknowledgments
A doctoral fellowship from the French Ministry o f Research and
Space is acknowledged for L . D . A . J . P . A . thanks Dr. S.W.
Provencher (EMBL, Heidelberg, Germany) and .R.G. Efremov
(Ovchinnikov and Shemyakin Institute, MoscoW,, Russia) for
providing us with useful source programs. Thanks are also due to
Prof. A.M. Tamburro (University o f Basilicata, Potenza) for his
support and helpful discussions.
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[2] Sage H., Gray W.R., Studies on the evolution of elastin. I.
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[41 Tamburro A.M., The elastin structure(s), in: Balduini C., Cheru-
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[10] Foster J.A., Bruenger E., Rubin L., Imberman M., Kagan H.,
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