Biochimica et Biophysica Acta, 492 (1977) 370-376

© Elsevier/North-Holland Biomedical Press

BBA 37685

C O N F O R M A T I O N A L T R A N S I T I O N S OF a-ELASTIN

A. M. TAMBURRO", V. GUANTIERI a, D. DAGA-GORDINI~ and G. ABATANGELO b

"lstituto di Chhnica Analitica and Centro di Studio sulla stabilitgl e reattivitgt dei composti di
coordinazione, C.N.R. and blstituto di lstologia ed Embriologia Generale, UniversiM di Padova (Italy)
(Received December 7th, 1976)

SUMMARY

The a-elastin, the soluble product obtained from elastin by oxalic acid hydro-
lysis, has been studied from a conformational point of view in different conditions
using circular dichroism spectroscopy.
The spectral variations of the a-elastin dissolved in water and in different
organic solvents/water mixtures have been examined in order to establish the effect of
the solvents on the structural properties of the protein.
The effect of calcium ions has also been studied on a-elastin dissolved either in
water or in organic solvents.
Finally, the effect of p H and temperature on the conformational properties of
the a-elastin has been investigated.

INTRODUCTION

a-Elastin, the soluble protein obtained from native elastin by oxalic acid hydro-
lysis, has necessarily been the object of the structural studies in solution which are im-
possible for the parent protein owing to its insolubility [1-4]. In addition, soluble
peptides, both crosslinked, and non crosslinked, have been studied from a conforma-
tional standpoint, mainly by circular dichroism (CD) spectroscopy [5-6]. Although
a-elastin is essentially unordered in aqueous solution, the appearance of an a-helix
structure has been shown in trifluoroethanol and water-ethanol mixtures [1-4]. This
could be of relevance in the correlation of the known elastic properties of this protein
with its conformational features. Furthermore, Urry [7-8] hypothesized the existence
in elastin of some specific conformations able to coordinate Ca z+ accounting for phe-
nomena such as atherosclerosis and calcification.
A particular aspect of this protein is the phase separation which occurs when an
aqueous solution of a-elastin is raised to 37 °C. The viscous, more dense phase is call-
ed the coacervate. The process depends on concentration and may be interpreted as
an intermolecular hydrophobic association [9].
It is the aim of the present paper to investigate, by CD, the influence of sol-
vent, temperature, p H and Ca z+ complexation on the ability of a-elastin to undergo
conformational transitions.
 371

MATERIALS AND METHODS

Preparation of purified elastin

Purified elastin was obtained from bovine ligamentum nuchae of l-2-year old
calves according to the established procedure [10]. The soluble a-elastin was obtained
from elastin by 0.25 M oxalic acid hydrolysis as described by Partridge et al. [10]. The
material represented 60 ~ of the original elastin. The amino acid composition of both
elastin and a-elastin was in agreement with reported values [i l].

Circular dichroism studies

C D measurements were obtained on a Cary model 61 dichrograph. Spectra
were taken at several temperatures between 8 and 80 °C controlled by a circulating
water bath. Temperatures were measured using a thermocouple contained in the sample
cell. Samples were equilibrated in the cell at a specificd temperature for at least 15
rain prior to analysis. Cylindrical quartz cells o f 0.1 and 0.05 cm path length were
employed. The data are expressed in terms o f the mean residue molar ellipticity in
units of deg- cm 2- d m o l - 1.

RESULTS

Solvent studies
Shown in Fig. 1 are the C D spectra of a-elastin at r o o m temperature under

i
O'
II j/.
et /
f
... i~ ~
0

/ / . ~

-3-

-4-

200 210 220 230 240 2540

~, (rim)

Fig. 1. CD spectra of a-elastin at room temperature in 80% ethylene glycol (------); water, pH 4.9
(------); water plus CaCl2 (1 mg/ml) (. . . . . . . ); 80% dioxane ( . . . . . . ); 80~ dioxane plus CaCl2
(I mg/ml) (A--A). The concentration of the protein was I mg/ml.
 372

different environments. A conformational transition is evident by changing the sol-

vent from pure water to 80 ~o dioxane, while intermediate forms seem to be present
in 80~o ethylene glycol. In aqueous solution the CD pattern has been interpreted [1]
as arising from the predominance of aperiodic structures with the possible presence of
small amounts of helical structures accounting for the shoulder at approx. 220 nm.
By decreasing the solvent polarity a tendency can be noted toward a single negative
band at approx. 225 nm, while the large negative band at shorter wavelengths (mainly
associated with the amide :r-Jr* transition of the aperiodic form)is substituted by a
region of positive dichroism. The effect of Ca 2+, which is almost negligeable in water,
seems to favour the conformational transition in organic solvents.
A detailed picture of the dioxane induced transitions at room temperature, in
the presence or absence of Ca 2+, is reported in Fig. 2. Apparently, multiple confor-
mational variations are evoked by the less polar solvent. At dioxane percentage up
to 40 ~ the spectra resemble those of the a-helix (two comparable negative bands at
about 220 and 210 nm), then the a-helix decreases and a new conformation appears at
higher concentrations of dioxane. This conformation is characterized by a negative
band at 223 nm and by at least one positive band at shorter wavelengths: these features
are reminescent of those recently predicted [12] for the so called [:Lbends [I 3-14]. The
effect of Ca z+ binding is to enhance the transition, apparently by stabilizing the f/-
bend as was suggested by Urry [7] and later found in soluble fragments of elastin [5].

-2

-3
/
4
S' o 2 0 °Io 40 % 50 % I 60 % 8O%
0

-2

-3

-4
20 % 40 % 50 % 60 % 80 %

200 220 240 200 220 240 200 220 240 200 22Q 240 200 220 240 200 220 240
(nrn)

Fig. 2. CD spectra of c~-elastin at room temperature in water-dioxane mixtures at the indicated

percentages of the organic solvent in the absence (upper part) and in the presence (lower part) of
CaCI2 (1 mg/ml). The concentration of the protein was 1 mg/ml.

Temperature studies
In Fig. 3 the influence of the temperature on the CD spectra of a-elastin in
water is shown. Clearly, the protein is not undergoing a simple two-state heat-induced
denaturation. Between 8 and 30 °C we observe a large reduction of both negative
bands with small shifts of the minima and no changing of the gross spectral features.
 373

±
-!

-3-

-6"

2bo 2{0 2~0 2~0 2,~0 250

?~(nm)

Fig. 3. CD spectra of a-elastin (1 mg/ml) in water, pH 4.9 at the indicated temperatures.

This hardly corresponds to a decrease of ordered secondary structure (e.g. a-helix) but
more probably reflects a change in the tertiary structure and/or a change in the degree
of intermolecular interaction. Although the conditions used were not such as to lead
to coacervation, aggregation cannot be excluded since the pH (4.9) was close to the
isoionic point of a-elastin where intermolecular interactions should be maximal.
There are indications that the ORD and CD spectra of aggregated helices of poly(e-
glutamic acid)[15] and mitochondrial structural protein [16] show small shifts and
significant variations in the magnitude of the extrema.
Between 30 and 80 °C only small changes are detectable: the negetive band at
approx. 205 nm decreases while the shoulder at longer wavelengths increases. Al-
though the variations are not unambiguously identifiable in terms of change into the
population of specific conformers, they are however compatible with an inverse
temperature transition with a small increase in ordered structure. In any case, it is of
major significance the lack of large dichroic changes revealing the disordering usually
associated with the thermal denaturation. The binding of calcium does not substantially
modify the trend exhibited by the protein in its absence: only minor variations were
detectable, and therefore the corresponding spectra are not reported.

pH studies
Fig. 4 reports the effect of pH on the CD spectrum of a-elastin. A conforma-
tional transition, associated with the titration of some ionizable groups, is observed.
However the results are not easily rationalizable in terms of change of known poly-
peptide secondary structure. As a matter of fact, on increasing the pH, the intensity
of both negative bands is enhanced: an increase of either a-helix or fl-structures (in-
cluding fl-bends) should diminish the band at shorter wavelengths, predominantly
associated with the aperiodic form, while an increase of the latter should diminish the
shoulder at longer wavelengths. Perhaps, a better explanation can at present be found
in a charge mediate effect, producing a freezing of a-elastin tertiary structure with con-
374

51
/, 0-8.0-9,0

-2

-3¸

~4.

-5-

o tY
6 . . . .

5 6 7 B 9pH

260 210 2~0 230 240 2&~o

(nm)
Fig. 4. C D spectra of ~L-elastin in 0.01 M T r i s . H C l buffer at the indicated pH values. In the insert
is reported the variation of the m e a n residue molecular ellipticity at the m i n i m u m o f the shorter
wavelengths negative band as a function of pH.

sequent enhancement of the optical activity. Of course, the possibility exists (not
necessarily as an alternative) that the increase of pH above 5 reduces the intermole-
cular interactions enhancing the solubility of the protein. It should be appreciated that
the spectral variations obtained by increasing the pH, are similar to those resulting
from a decrease of the temperature below 30 °C (Fig. 3).
The pH-induced changes are almost abolished in the presence of CaCI2 under
saturing conditions. Probably, at least some C a 2+ would interact with the ionizing
groups of the protein responsible for the above mentioned spectral variations obtained
in the absence of Ca z+.

DISCUSSION

In discussing the properties of a-elastin in solution, it should be appreciated

that the fully hydrated protein is probably not a satisfactory model for the native in-
soluble elastin. It is interesting to note that fibrous elastin is a more ideal elastomer in
dimethylsulfoxide or ethylene glycol than in water [17] and therefore the conforma-
tion of a-elastin in the above mentioned organic solvents is conceivably more re-
levant to fibrous elastin, as recently emphasized by Urry and Long [18]. Accordingly,
our studies in water/ethylene glycol and water/dioxane mixtures reserve particular
comments. An important point is that a-elastin is not devoid of order in these sol-
vents but assumes different conformations such as the Ct-helix and the B-bend. Ac-
 375

cording to Sandberg et al. [19] and Urry [20], candidates for the former structure are
the alanine rich regions associated with cross-links, while the repeating sequences
Val-Pro-Gly-Gly, Val-Pro-Gly-Val-Gly and Ala-Pro-Gly-Val-Gly-Val are likely to
contain fl-bends. These segments of secondary structure are probably present, al-
though in less amounts (and therefore masked by the predominant random conforma-
tions), also in water. On increasing the temperature of aqueous solutions of a-elastin
up to about 30 °C it is probable that changes in the tertiary structure and hydrophobic
aggregation occur, the two phenomena being connected. Higher temperatures do not
substantially affect the aggregate, apart from a small increase or order as judged by
the decrease and the red shift of the shorter wavelength band and by the enhancement
of the shoulder (Fig. 3). As in the case of solvent-induced transitions, the spectra are
compatible with the appearance of both a-helix and fl-bends. The process here de-
scribed refers to inter- and intramolecular interactions evoking conformational
changes in homogeneous phase, but it is immediate in its extension to the intermolecular
association of a-elastin to produce the coacervate. In this context our results are in
agreement with the suggestion of Urry and coworkers [4, 18, 20] about the ordered
regions of elastin consisting of a sequential arrangement of fl-spiral (repeating fl-bends)
and a-helical segments along a single chain. A concentration dependent association of
the chains through hydrophobic interactions produces coacervation of a-elastin with
resulting fiber formation [19, 21]. The structural element for such interaction mechan-
ism would be provided by the alignement of spiralling hydrophobic ridges of two
associating/3-spirals, resulting in the twisted rope structures seen by electron micro-
scopy [22].
The last point to be discussed is the effect of Ca 2+. As anticipated in the intro-
duction, Urry [7] proposed that Ca z+ binds at neutral sites. In this model, the acyl
oxygens of the peptide moiety are directed nearly perpendicular to the plane of the
other atoms in a fl-bend, and therefore in an exposed position favourable for coordina-
tion of the ions. Experimental support came later from studies on a-elastin in tri-
fluoroethanol [4, 8] and on the coacervate [23-26]. In particular it was shown that
calcium effects a notable conformational change on a-elastin in trifluoroethanol and
it is still able to bind (as judged by infra red and electron microscopy) to the N-formyl-
O-methyl ester derivative of a-elastin coacervate, where the charged groups were
blocked. On the other hand it has been shown, using both water suspension of elastin
[27] and aqueous solution of a-elastin [11] that the binding involves very small amount
of Ca 2+, but it is strongly pH dependent. This would alternatively suggest binding at
charged sites, possibly carboxyl groups.
Our results on a-elastin on this point are not conclusive. The binding of Ca 2÷
seems to favour the conformational transition toward a specific conformation, i.e. the
/3-bend in dioxane, without affecting, however, the changes evoked by the tempera-
ture. On the other hand, the large dichroic changes induced by the pH, are undoubted-
ly related to ionizing groups, and are almost abolished by saturation of Ca -'+ . This
would indicate binding to charged sites. It is tempting to suggest that both carboxyls
and acyl oxygens are able to bind calcium in a-elastin, the charged groups being the
preferred sites of binding in molecularly dispersed aqueous solutions, while the neutral,
dipolar groups become increasingly important in organic solvents and in the coacer-
vate, in which the ionization of carboxyl group is conceivable more difficult. In native
elastin probably the situation is intermediate in that both mechanisms are acting.
376

ACKNOWLEDGEMENTS

W e are g r a t e f u l l y i n d e b t e d to Prof. L o r e n z o G o t t e for helpful discussion.

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