(Received for publication, July 13, 1995; and in revised form, August 17, 1995)
From the
Elastin is the macromolecular polymer of tropoelastin molecules
responsible for the elastic properties of tissues. The understanding of
its specific elasticity is uncertain because its structure is still
unknown. Here, we report the first experimental quantitative
determination of bovine elastin secondary structures as well as those
of its corresponding soluble -elastin. Using circular dichroism
and Fourier transform infrared and near infrared Fourier transform
Raman spectroscopic data, we estimated the secondary structure contents
of elastin to be
10%
-helices,
45%
-sheets, and
45% undefined conformations. These values were very close to those
we had previously determined for the free monomeric tropoelastin
molecule, suggesting thus that elastin would be constituted of a
closely packed assembly of globular
structural class tropoelastin
molecules cross-linked to form the elastic network (liquid drop model
of elastin architecture). The presence of a strong hydration shell is
demonstrated for elastin, and its possible contribution to elasticity
is discussed.
The elasticity required for the appropriate functioning of skin, lung, and large blood vessels is due to the presence of elastic fibers within their extracellular matrix(1) . The predominant component of these complex structures is the elastin protein, which endows them with its characteristic property of elastic recoil. Elastin is a macropolymeric protein synthesized by mesenchymal cells as a soluble precursor, tropoelastin, whose primary transcript undergoes alternative splicing resulting in the translation of several protein isoforms(2, 3) . After release in the extracellular space, most of the lysyl residues of tropoelastin are enzymatically deaminated. Following a series of non-enzymatic reactions, the activated residues condense to form specific tetrafunctional cross-links, named desmosines, which appearance allows the spreading of the elastic network within the microfibrillar component of the fiber (for a review, see (1) ).
The primary structure of BTE ()(3, 4) consists of an alternance of
cross-linking regions, where the lysyl residues are located, and of
large hydrophobic domains responsible for elastin elasticity. The
highly hydrophobic BTE molecule possesses a very basic C-terminal
sequence where its only two Cys residues are located. Those were
recently shown to form an intrachain disulfide bridge stabilizing an
hydrophilic pocket(5) . This C-terminal feature seems involved
in elastin fiber assembly(6) .
The presence of numerous cross-links and the extreme hydrophobicity of BTE chains are responsible for the great resistance of polymeric BE as well as its total insolubility in any solvent(1) . BE-K is the heterogeneous mixture of peptides obtained from BE when it is solubilized by KOH(7) . It is a form more suitable for biological tests than BE, as it is soluble. BE-K is thought to be a good model of insoluble BE because of its ability to form a matrix (coacervate) akin to hydrated insoluble BE, the elastic form of BE, at physiological temperatures and high concentrations(7) .
The elasticity of BE has an entropic nature(8) . However, its exact origin remains uncertain, as the results gathered about BE and BE-K structures are few. Indeed, the very peculiar physico-chemical properties of these molecules do not allow significant structural results using the classical physical investigation methods. This lack of structural data explains why BE molecular models forward an explanation of the elastic mechanism without knowledge of its structures. A description of BE conformation is urgently needed.
Among the various models proposed (see (1) for a review),
only three are still discussed. (a) In the globular liquid
drop model of Weis-Fogh and Andersen(9, 10) , BE is
described as an aggregate of tropoelastin globules. Elasticity
originates from hydrophobic interactions at the protein solvent
interface of the globules as they deform during stretching. (b) The random network (8) regards BE as a protein
devoid of any organization, much as rubber. It is connected to a
classical elasticity theory. This model is supported by the works of
Tamburro and co-worker (11, 12) who have established
that peptides found in BTE sequence form transient -turns, which
stability is influenced by both the surrounding water (13, 14) and the length of the peptide(15) . (c) In the fibrillar
-spiral model of
Urry(16, 17) , BE is considered as a regular
arrangement of consecutive
-turns (
-spiral). In this context,
elasticity arises from librational motions at the level of the spiral
-turns.
Following predictive (18) and experimental (19) evidences, we have proposed a -class molecular model
for BTE. The present work reports the structural investigation of the
polymeric forms of this molecule, BE and BE-K, using CD, FT-IR, and NIR
FT-R spectroscopies. The first estimations of BE and BE-K secondary
structure contents are presented. The numerical values obtained for the
polymers are compared to those formerly determined for monomeric BTE.
Their consequences toward BE existing models and the possible
elasticity mechanisms are discussed.
Secondary structures were determined by
decomposition of the amide I band (C=O stretching mode of the
peptidic bond, 1630-1700 cm) into individual
components assigned to substructures, as the Raman sensitivity of that
band to conformation is well
known(23, 24, 25) . First, Fourier
self-deconvolution(26) , second derivative(27) , and
maximum entropy (28) methods were independently applied to the
original amide I bands. Second, among the components yielded by the
resolution enhancement methods, only the positions of the most
conserved and prominent ones were used as input parameters for a least
square curve-fit procedure. No parameters were fixed during the
calculation except the nature of the underlying profiles, which were
assumed to be 80% gaussian and 20% lorentzian. The structural
assignments of the computed components were made according to both
their positions before and after
reconstruction(23, 24, 25) . The cumulated
fractional area contribution assigned to a given substructure
represented its relative total content in the protein conformation. The
enhanced profiles were computed by the SPOV program (developed in
Ovtchinnikov and Shemyakin Institute in Moscow). The decompositions
were made using the CURVEFIT module of the LabCalc package (Galactics
Industries).
For side chains such as alanine, valine, leucine, and
isoleucine, the most prominent frequencies are those associated with
the CH bending mode found at 1465 ± 20
cm
(29) and with the CH
antisymmetric deformation mode found at 1450 ± 20
cm
(22) . The behavior of the band centered
around 940 cm
has been studied, as it is
characteristic of the ordered
-helices as shown for
poly-L-lysine used as a model(30, 31) . Bands
arising from aromatic (tyrosine, phenylalanine) and sulfur (cysteine,
methionine) residues and from the polypeptide backbone were readily
identified(32, 33) .
The amino acid composition of BE (Table 1) was in good agreement with those obtained by others(7, 20) . The preparation was free of collagen as the level of Hyp residues was low, and no hydroxylysine was detected. Likewise, the presence of Asp and Glu residues with values that compared to BTE Asp and Gln ones, respectively, demonstrated the absence of microfibrillar proteins. Fundamentally, no Trp nor His was detected, and the estimated quantities of Gly, Ala, Pro + Hyp, Val, Leu, and Ile residues compared well with those of BTE composition (Table 1). The main discrepancy between elastin and BTE compositions was the estimated number of lysyl residues. This arose from the great difficulty in detection of all elastin cross-linking amino acids. With the occurrence of one Met residue being below the technique precision, the composition of BE (Table 1) indicated a high level of purity, allowing its solubilization and the use of optical spectroscopic methods to analyze its structures.
The main features observed in the FT-IR spectra of proteins are those associated with the planar peptidic bond vibrational modes, the so-called amide bands, which positions, widths, and intensities are characteristic of the vibrational modes associated and thus of the local geometry of the peptidic chain. They are the amide I (C=O stretching), amide II (mainly C-N stretching), amide III (N-H in plane deformation), and amide A (N-H stretching) bands; the first three are very sensitive to conformational changes(34, 35) , while the last one brings information about the hydrogen bondings undergone by the peptidic N-H groups(22) .
The FT-IR spectra of our samples in
KBr pellets (Fig. 1) compared well with the data obtained by
others for insoluble elastin (36) and solubilized
elastin(37) . The two molecules shared close global
conformations as their structure-sensitive amide I, II, and III bands
were found at comparable positions (amide I at 1659 and 1657
cm, amide II at 1538 and 1542 cm
,
and amide III at 1237 and 1238 cm
, for BE and BE-K,
respectively). However, BE amide I band was much broader than that of
BE-K, underlining some structural differences. The occurrence of amide
A bands at different positions (3322 and 3307 cm
,
respectively) also demonstrated that their peptidic N-H groups
were involved in different types of hydrogen bondings.
Figure 1: FT-IR spectra of bovine elastin KBr pellets. upper, BE; lower, BE-K. Only the positions of the amide bands are marked.
Laser-visible Raman spectroscopy is a very powerful technique to investigate the conformation of biological molecules, as it can provide information about the secondary structures, the microenvironment of the residues, and the polypeptidic backbone geometry. However, BE is so highly fluorescent that convenient standard Raman data could not be reached(38, 39) . Thus, we have preferred NIR FT-R spectroscopy instead of normal visible Raman, as the use of an infrared source was less likely to excite the intense protein autofluorescence.
The characteristic bands of a protein Raman spectrum were observed
in the BE spectrum (Fig. 2) as follows: 1) the conformationally
sensitive amide I (C=O stretch, 1630-1700
cm) and amide III bands (N-H in plane
deformation, 1230-1310 cm
), which arise from
the Raman-active vibrational modes of the planar CONH peptidic bond; 2)
bands assigned to residue side chains like aromatic cycles (only Phe
and Tyr in the present case), CH, CH
, and CH
groups, and stretching of bonds containing sulfur atoms; and 3)
bands corresponding to the C
-C and
C
-N stretches of the polypeptidic backbone. The
existence of disulfide bridges in BE was clearly demonstrated by the
occurrence of two bands assigned to S-S (527
cm
) and C-S (665 cm
)
stretching modes, respectively. These should mainly possess a local
gauche-gauche-trans-geometry, as the S-S mode was observed at 525
± 10 cm
(32) . They certainly arose
from BTE intrachain bondings. The NIR FT-R spectrum of BE-K, in
contrast to the BE spectrum, showed a relatively poor signal-to-noise
ratio reflecting the very heterogenous nature of the solubilized
elastin (data not shown). Nevertheless, its amide I band was clear
enough for structural analysis.
Figure 2:
NIR
FT-R spectrum of BE in powder in the frequency region 500-1750
cm and proposed assignments.
,
stretching;
, deformation; G-G-T,
gauche-gauche-trans; (
), band at 934 cm
corresponding the C
-C stretching mode of
-helices(30, 31) ; (
), amide III
band maximum at 1248 cm
in the frequency domain of
-structures (24) .
Our analysis of the Raman data has
mainly been focused on secondary structure quantitation. The amide I
band originates from the C=O stretching modes of all the
peptidic bonds of the protein. Depending upon the particular secondary
structure, a C=O group is involved in a given type of hydrogen
bonding, whose characteristics influence its frequency of vibration.
That way, all the C=O occurring in -helices will vibrate
similarly but differently from those encountered in
-sheets.
Likewise, regular
-sheets and irregular ones will yield different
signals. The vibrational frequencies from one substructure to another
are not very different but sufficient to be distinguished(22) .
They all occur in the same characteristic spectral range
(1630-1700 cm
), and their respective Raman
signals overlap to yield the complex amide I band. Decomposition
methods aim at directly accessing those structural contributions that
overlap. In this manner, it is thereafter possible to determine the
secondary structure contents of the molecule by the standard assumption
that their respective areas correspond to their conformational
contributions(23, 24, 25) . The mathematical
solution to a given decomposition problem is never unique, and it is
always very difficult to tell which solution is the best, as one has no
idea of how many contributions really exist and where they are located.
But, fortunately, the use of more than only one resolution enhancement
method permits assessment of these parameters. Here, we have used the
three major ones. Each of them proposed several underlying
contributions in our amide I profiles (data not shown). By comparison
and correlation between their results, we were able to choose a small
representative number of components whose positions could be accurately
estimated. The calculated components centered in
1630-1700-cm
range were assigned to
-helices,
-strands, and undefined (turns + coils)
secondary structure elements according to both theoretical and
experimental
results(22, 23, 24, 25) .
A
component near 1640 cm corresponding to the
hydration water bending mode (32) was resolved in both
decompositions (Fig. 3, Table 2) as the molecules were in
the solid state. This result was in good agreement with the paradoxical
demonstration that the very hydrophobic and insoluble BE formed very
tight hydrogen bondings with water(40) . One helix, two
-strands, and two undefined components were evidenced for either
BE (Fig. 3a) or BE-K (Fig. 3b). The
occurrence of an
-helical component correlated well with the
observation of a band centered around 934 cm
in the
BE spectrum (Fig. 2), as that feature is characteristic of
ordered helices(30, 31) . Moreover, the presence of
-strands was confirmed by the position of BE amide III band (1248
cm
) since it fell within the typical Raman amide III
domain of
-structures (24) .
Figure 3:
Decompositions of the NIR FT-R amide I
bands of bovine elastins. a, BE; b, BE-K. The
experimental (solid line) and calculated (dashed
line) profiles were compared. The components computed in the
1630-1700-cm amide I domain have been assigned
as follows: HW, hydration water;
,
-helices;
,
-strands; U, undefined
conformations (turn + coil). Those corresponding to secondary
structure elements were represented by a solid line. Their
parameters are compiled in Table 2and were used for quantitation (Table 3).
The quantitative results
compiled in Table 3showed that BE and BE-K possessed similar
global conformations, which were consistent with high levels of both
extended (43 and 46%, respectively) and unordered (48 and 41%,
respectively) structures. However, the analysis of their respective NIR
FT-R amide I components (Fig. 3, Table 2) indicated strong
variations in their local structures. For example, the first undefined
structure component (1661 cm for BE and 1656
cm
for BE-K; see Table 2) accounted for 45% of
the global structure before solubilization and 12% after. Meanwhile,
the second undefined structure component (1672 cm
for BE and 1666 cm
for BE-K; see Table 2) varied from 3 to 29%. So, an inversion of population
between the modes giving rise to those components had occurred, and the
conditions of hydrogen bondings were different between the two
molecules as revealed by the FT-IR data analysis. Insoluble and soluble
elastins had quantitatively but not qualitatively identical
conformations. This observation strongly suggests that BE-K is probably
not a good model for BE, as far as local conformations are concerned.
The CD spectrum of BE-K in water (Fig. 4) was in good
agreement with those recorded previously by others for soluble elastins (41, 42) . The broad negative band observed at 200 nm
tended to support the view that BE-K was disordered. However, this
spectral feature could be assigned to short and distorted -sheets (43) as was the case for the BTE spectrum(19) . The CD
quantitation (Table 3) confirmed this possibility, as a high
level of
-structures was estimated for BE-K disolved in water. The
value (47%) compared well with that estimated for BE-K in the solid
state (46%). Nevertheless, the structural contents of BE-K seemed to
change when it was dissolved (Table 3). The most striking feature
was the apparent disappearance of
-helical structures. This was
quite surprising, as BTE helices (
5%) were preserved in
solution(19) . A hydration effect upon BE-K helices remained
possible but uncertain all the more so since the precision of the
quantitative methods used was ±5%.
Figure 4: CD spectrum of BE-K solubilized in water.
The conformation contents
of BE (Table 3) were in very good agreement with those of our
free BTE -class molecular model(19) . For BTE, they were
5% of helices (cross-linking domains), 50% of
-strands, and 45% of
undefined conformations (rest of the molecule or elastic regions). The
finding that the global structures of free and cross-linked BTE were
significantly identical strongly suggested that our monomeric model
could apply to the elastin polymer, meaning that BE would be
constituted of a three-dimensional arrangement of globular BTE
molecules connected by cross-links. This structural definition
typically corresponded to the liquid drop model of
BE(9, 10) . This model was also greatly supported by
recent scanning tunneling microscopy observations of reconstituted BE (44) and human recombinant tropoelastin coacervates. (
)Our structural results clearly contradicted the random
network (8) and
-spiral (16, 17) models
of BE architecture, as BE did possess high levels of ordered
structures.
The structure of BE could thus be described as a
three-dimensional repetition of our molecular model of
BTE(19) , which is to say that -class BTE molecules are
closely packed together and cross-linked by helical domains (
10%
-helices), while the entropic ``elastic'' regions would
consist mainly of buried short and/or distorted antiparallel
-strands (
45%), which are probably packed in
-barrels
and alternate with external turns and coil substructures (
45% for
the sum). In addition, we would like to underline that the hydrophobic
domains of BE are highly mobile (45, 46) and that
coil-turn (11, 12, 15) or sheet-coil-turn (18) conformational transitions are possible. These transitions
are most certainly mediated by the hydration water
molecules(13, 14, 18) .
The present work reports the first experimental estimation of the secondary structures of insoluble bovine elastin. Conclusions about the tertiary and quaternary structures of the elastomer have also been reached. Our results provide valuable information for understanding the elastic function of BE as they demonstrate that the structure-elasticity relationships must be envisaged in a liquid drop architecture context(9, 10) .
Nevertheless, we point out that our results do not mean that the elasticity mechanism (hydrophobic interactions) proposed in 1970 by Weis-Fogh and Andersen (9, 10) is correct. We only agree with the architecture they have proposed for the molecule. Indeed, we suggest that their explanation of elasticity is incorrect, as it is based on a diphasic description of the swollen polymer (protein chains + water) and neglects the hydration water of the molecule.
Recently, water solvent was shown to act as a plasticizer for elastin (47) , that is to say it enhances its mobility. Moreover, the action of solutes on the structure of elastin is indirect and seems to be mediated through its hydration shell(48) . That way, if solvent water molecules are considered as particular solutes, their plasticizing effect should be processed through the hydration shell of the molecule. We thus feel that the strong hydration shell demonstrated for elastin could have some functional significance. Swollen elastin would then be better described as a triphasic system, protein + hydration water + solvent water, and, in this view, the conformational transitions we and others have proposed for BE hydrophobic domains (11, 12, 15, 18) should have a functional role. The elasticity theory connected to this proposal and the molecular events occurring during stretching or relaxation need now to be completely described. Further experiments in this way are in hand as well as molecular modelings of isolated and/or cross-linked tropoelastins.