Disulphide structure of a sunflower seed albumin: conserved and variant disulphide bonds in the cereal prolamin superfamily.
ABSTRACT Disulphide mapping of a methionine-rich 2S albumin from sunflower seeds showed four intra-chain disulphide bonds which are homologous with those in a related heterodimeric albumin from lupin seeds (conglutin delta). Similar conserved disulphide bonds are also present in alpha-gliadin and gamma-gliadin storage proteins of wheat, but a lower level of conservation is present in a further related group of proteins, the cereal inhibitors of alpha-amylase and trypsin. These differences may relate to the different functions of the proteins.
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ABSTRACT: 2S albumin fractions were isolated by a modified acetone precipitationmethod (Kortt and Caldwell 1990) from seeds of 103 sunflower (Helianthus annuus L.) accessions and analysed by SDS-PAGE, IEF andRP-HPLC. Two methionine-rich albumins SFA7 and SFA8 showed nodifferences in mobility on SDS-PAGE gels but were readily separated byRP-HPLC. Their levels also varied widely between different genotypes, inrelation to each other and as proportions of the total albumin fraction. Avariant form of SFA8 was identified which differed from the normal SFA8in its pI (6.5 compared to 6.0) and mobility on SDS-PAGE. N-terminal sequences of both the variant form of SFA8 and the majorform of SFA7 were identical to that reported previously for the normalform of SFA8 from the cultivar Hysun (Kortt et al., 1991) indicating theirstructural relatedness. Analysis of segregation in the F2 of the crossbetween lines VIR130 (variant SFA8) and VIR104 (normal SFA8) showedthat the normal and variant forms of SFA8 are encoded by alleles at asingle Mendelian locus. The levels of SFA7 and SFA8 in the seeds ofparental lines, F1 hybrids and individual F2 seeds classifiedfrom SDS-PAGE and IEF as homozygous for normal SFA8 (VIR104 type),homozygous for variant SFA8 (VIR130 type) and heterozygous (F1type) were determined by RP-HPLC. Seeds of the parental line VIR130contained 3.7% SFA7 and 19.0% SFA8 whereas seeds of VIR104contained 9.9% SFA7 and 12.8% SFA8. The F1 hybrid seedscontained a higher total amount of SFA7+8 proteins (32% comparingto 22% in each parent) which was largely accounted for by a highproportion of SFA7. The mean combined proportions of SFA7+8 in eachof the three phenotypic classes of F2 seeds were about 18–19% ofthe total. However, the combined proportions of SFA7+8 varied in therange 10–20% among the individual seeds. The ratio of SFA7 to SFA8was highest in the VIR104-type and heterozygous seeds, with the amountof SFA7 exceeding that of SFA8 in six heterozygous seeds. Theproportions of SFA7 and SFA8 were inversely correlated among individualF2 seeds. The results suggest that the amounts and proportions ofSFA7 and SFA8 are determined by genetic factors in addition toavailability of sulphur.Euphytica 12/2002; 129(1):99-107. · 1.64 Impact Factor
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ABSTRACT: Among cereals, Avena sativa is characterized by an extremely soft endosperm texture, which leads to some negative agronomic and technological traits. On the basis of the well-known softening effect of puroindolines in wheat kernel texture, in this study, indolines and their encoding genes are investigated in Avena species at different ploidy levels. Three novel 14 kDa proteins, showing a central hydrophobic domain with four tryptophan residues and here named vromindoline (VIN)-1,2 and 3, were identified. Each VIN protein in diploid oat species was found to be synthesized by a single Vin gene whereas, in hexaploid A. sativa, three Vin-1, three Vin-2 and two Vin-3 genes coding for VIN-1, VIN-2 and VIN-3, respectively, were described and assigned to the A, C or D genomes based on similarity to their counterparts in diploid species. Expression of oat vromindoline transgenes in the extra-hard durum wheat led to accumulation of vromindolines in the endosperm and caused an approximate 50 % reduction of grain hardness, suggesting a central role for vromindolines in causing the extra-soft texture of oat grain. Further, hexaploid oats showed three orthologous genes coding for avenoindolines A and B, with five or three tryptophan residues, respectively, but very low amounts of avenoindolines were found in mature kernels. The present results identify a novel protein family affecting cereal kernel texture and would further elucidate the phylogenetic evolution of Avena genus.Molecular Genetics and Genomics 08/2014; · 2.88 Impact Factor
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ABSTRACT: Alignment of 98 S-rich prolamin amino acid sequences illustrated that four cysteines were preserved nearly 100% and four cysteines were less well conserved. These residues form two to four intramolecular disulfide bonds in several S-rich prolamins. For maize gamma zein, it is unknown whether the eight C-terminal cysteines form intramolecular disulfide bonds. If gamma zein contains intramolecular disulfide bonds, modifications of critical cysteines in soluble thioredoxin–gamma zein fusion protein might prevent disulfide bond formation and result in misfolded and insoluble protein in Escherichia coli. Individual modification of conserved cysteines C128 and C136 resulted in a four-fold reduction in solubility and a significant decrease in expression level compared to wild-type fusion protein. Modification of conserved C156 resulted in a two-fold reduction in expression level but not a significant change in solubility until combined with a non-deleterious Q181R modification. This suggested that C128 and C136 were involved in disulfide bonds critical for protein folding, whereas C156 was more critical for protein stability. Modification of a non-conserved N-terminal cysteine residue (C117) resulted in increased protein solubility, suggesting it was not involved in an intramolecular disulfide bond. From these data and a review of the literature, a disulfide map for gamma zein is proposed.Plant Science. 01/2002;
FEBS 17723 FEBS Letters 396 (1996) 285 288
Disulphide structure of a sunflower seed albumin: conserved and variant
disulphide bonds in the cereal prolamin superfamily
T.A. Egorov ~, T.I. Odintsova b, A.Kh. Musolyamov ~, R. Fido c, A.S. Tatham c, P.R. Shewry c,*
~Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., Moscow 117984, Russian Federation
~' Vavilov Institute of General Genetics, Russian Academy of Sciences, 3 Gubkina Str., Moscow 117809, Russian Federation
~IA CR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BSI8 9AF, UK
Received 2 September 1996; revised version received 20 September 1996
Abstract Disulphide mapping of a methionine-rich 2S albumin
from sunflower seeds showed four intra-chain disulphide bonds
which are homologous with those in a related heterodimeric
albumin from lupin seeds (conglutin 5). Similar conserved
disulphide bonds are also present in c~-gliadin and T-gliadin
storage proteins of wheat, but a lower level of conservation is
present in a further related group of proteins, the cereal
inhibitors of c~-amylase and trypsin. These differences may
relate to the different functions of the proteins.
Key words." Seed; Cereal; Sunflower; Wheat; Albumin ;
Inhibitor; Gliadin; Disulfide bond
The cereal prolamin superfamily of seed proteins comprises
three major groups of proteins: the storage prolamins of bar-
ley, wheat and rye, the cereal inhibitors of m-amylase and
trypsin and 2S albumin storage proteins present in seeds of
a range of dicotyledonous plants [1 3]. Although these three
groups of proteins differ widely in their properties (including
Mr and amino acid sequences) they are characterized by the
presence of conserved cysteine residues. This conservation is
assumed to reflect a crucial role of these cysteine residues in
stabilizing the protein structure via disulphide bond forma-
tion, but this remains to be established as few components
have been disulphide mapped, and no detailed comparisons
have been reported.
2S albumin storage proteins have been characterized from a
diverse range of plants, including legumes (lupins, peas), cru-
cifers (radish, oilseed rape, Arabidopsis), cotton, sunflower,
Brazil nut and castor bean . These are typically heterodi-
meric proteins, with large and small subunits of Mr about
9000 and 4000, respectively, which arise from post-transla-
tional proteolysis of a precursor protein. One such component
has been disulphide mapped, the conglutin 5 of lupin. How-
ever, post-translational proteolysis does not occur in sun-
flower, and the albumin fraction consists of monomeric pro-
teins of Mr about 10000 18000 [5,6]. We have therefore
determined the disulphide structure of one such sunflower
albumin, a methionine-rich component SFA8 . The pattern
of disulphide bonds is identical to that in the heterodimeric
conglutin 5  and comparison with structures reported for
members of the prolamin and inhibitor groups shows the pres-
ence of conserved and variant disulphide bonds in this super-
family of seed proteins.
*Corresponding author: Fax: +44 (0)1275 394299.
0014-5793/96l$12.00 © 1996 Federation of European Biochemical Societies.
PHS0014-5793(96)01 1 17-9
2. Materials and methods
5 M cyanogen bromide in acetonitrile was obtained from Aldrich.
Guanidine hydrochloride and Tris were obtained from Sigma. Se-
quence grade trifluoroacetic acid (TFA) was from Applied Biosystems
and HPLC grade acetonitrile from Merck. Water was purified using a
Milli Q System (Millipore). All other reagents were analytical grade.
2.2. Protein purification
SFA8 was purified from sunflower Hybrid 246 as described by 
with an additional final separation by RP-HPLC on a SynChropak
RP-P C18 Semi-preparative column (10X 250 ram) with a linear gra-
dient (25% to 55%) of acetonitrile containing 0.07% (v/v) TFA in
0.05% (v/v) aqueous TFA.
2.3. Cyanogen bromide cleavage
0.5 mg (--40 nmol) of purified SFA8 was dissolved in 100 I.tl of 70%
(v/v) TFA and 25 I11 of 5 M CNBr in acetonitrile (about 200 molar
excess over methionine residues) added. The reaction mixture was
flushed with argon and incubated at 22°C for 18 h in the dark. After
drying the peptides were dissolved in 50 pl of 5 M guanidine hydro-
chloride in 0.1% (v/v) TFA and separated by RP-HPLC using an
Aquapore RP-300 C8 column (4.6 x 220 mm) with a linear gradient
(0 to 40%) of acetonitrile containing 0.08°/,, (v/v) TFA in 0.1% (v/v)
2.4. Identification of disulphide-bonded peptides
About 10% of each dried fraction was redissolved in 50 ~tl of 0.1 M
Tris-HC1 buffer, pH 8, containing 5 mM EDTA and 5 M guanidine
hydrochloride. Reduction of disulphide bonds and alkylation of sul-
phydryl groups was carried out by adding 1.4 ~tmol of dithiothreitol
dissolved in 1 ~tl of distilled water, standing for 30 min at 22°C, and
adding 1 ~tl (9.3 pznol) of 4-vinylpyridine. The reaction mixture was
then separated by RP-HPLC as described for the cyanogen bromide
digest, in order to identify fractions giving two peaks.
2.5. N-terminal amino acid sequencing
N-terminal amino acid sequence analysis was carried out with a
Model 816 protein sequencer (Knauer, Berlin) equipped with a Model
120A PTH Analyser (Applied Biosystems) operated according to the
manufacturer's instructions. Peptide samples were applied onto Im-
mobilon membrane in 30% (v/v) acetonitrile containing 0.1% (v/v)
N-terminal amino acid sequencing of the SFA8 fraction
purified from sunflower Hybrid 246 showed that it was iden-
tical for 20 residues to the sequence reported by Kortt et al.
. The latter showed the presence of eight cysteine residues
and 16 methionines in a protein of 103 residues (Fig. 1A).
With the exception of two adjacent residues (Cys-51 and
Cys-52), all the cysteines are separated by at least one methio-
nine. The purified protein was therefore cleaved with cyano-
gen bromide and 11 fractions were purified by RP-HPLC
(Fig. 2). Re-separation of each fraction after reduction and
All rights reserved.
ZA. Egorov et al./FEBS Letters 396 (1996) 285~88
A PROTEIN SEQUENCE
B PEPTIDE SEQUENCES
87AHNL P I ECNLM 97
C DISULPEIDE MAP
ii 24 51 52 62 64 94 I01 1
Fig. 1. Disulphide bond mapping of the sunflower albumin SFA8. (A) The protein sequence reported by Kortt et al. . (B) The sequences of
disulphide-bonded peptides isolated as fractions 4, 7 and 11. (C) Disulphide map of the protein.
alkylation showed that three of them (nos. 4, 7, 11) consisted
of two or more peptides linked by disulphide bonds. These
fractions were therefore subjected to N-terminal sequence
analysis, both before and after reduction of disulphide bonds.
This showed that fraction 4 consisted of two peptides linked
by a disulphide bond between Cys-64 and Cys-101 and frac-
tion 7 of two peptides with a disulphide bond between Cys-11
and Cys-62. Fraction 11 consisted of three peptides with
Cys-24 and Cys-94 linked to the adjacent cysteine residues
Cys-51 and Cys-52, but it was impossible to identify the pre-
cise bonds involving these two residues (Fig. 1B). The disul-
phide structure of SFA8 was therefore established as shown in
The disulphide structure of one heterodimeric 2S albumin,
namely conglutin 8 from lupin which has nine cysteines, has
T.A. Egorov et aL/FEBS Letters 396 (1996) 285-288
/0 20 30 40
Fig. 2. Separation of products of cyanogen bromide cleavage of intact SFA8 (approx. 0.5 mg) by reversed-phase HPLC on an Aquapore RP-
300, C8 column (4.6 × 220 ram). Peptides were eluted with a linear gradient of acetonitrile in the presence to TFA (from 0% B to 40% B for 90
min) at a flow rate of 0.5 ml/min. Solvent A: 0.1% (v/v) aqueous TFA; solvent B: acetonitrile containing, 0.08% (v/v) TFA. Fractions 4, 7 and
11 contained disulphide-bonded peptides.
been reported . Despite a low level of overall sequence
identity to SFA8 (17% homology), eight of the cysteine resi-
dues in conglutin 8 are clearly homologous, based on position
and sequence context, to those in SFA8 (Fig. 3). These cys-
teine residues, called A-H in Fig. 3, also form the same 4
disulphide bonds, two of which are inter-chain in conglutin
8. In neither case was it possible to discriminate between the
bonds involving the adjacent cysteine residues (C and D). The
ninth cysteine residue in conglutin 8, Cys-45, is not present in
SFA8 and is unpaired. This is called Cys J in Fig. 3.
Six of the eight cysteine residues present in SFA8 and con-
glutin 8 (cysteines B, C, D, F, G, H) are also conserved in the
C-terminal domains of two types of monomeric wheat prola-
min, called c~- and [3-gliadins (Fig. 3). These six conserved
cysteine residues also form the same three disulphide bonds
in the ~x- and [3-gliadins as in the 2S albumins (B to C or D, C
or D to G, F to H), while the 7-gliadin also contains an
additional pair of cysteine residues (called K and L in Fig.
3) which form a fourth disulphide bond [9-11].
The disulphide structures of three cereal cx-amylase/trypsin
inhibitors have been reported: the monomeric 0.28 (WAI-
0.28)  and tetrameric 0.53 (WAI-0.53)  (x-amylase in-
hibitors from wheat (Fig. 3), and a bifunctional c~-amylase/
trypsin inhibitor (RBI) from ragi (Indian finger millet) 
(not shown). Again it is possible to identify conserved cysteine
residues homologous to those present in the 2S albumins and
the gliadins, but the patterns of disulphide bond formation are
WAI-0.28 and RBI both have 10 cysteine residues, corre-
sponding to cysteines A to H and K in the gliadins and the 2S
albumins and a single unique cysteine residue called M. The
disulphide maps of these proteins were determined using
NMR spectroscopy (RBI) and mass spectroscopy (WAI-
0.28), showing identical patterns. Also, in both cases, it was
possible to distinguish between disulphide bonds involving the
adjacent cysteine residues (C and D), in contrast to the other
proteins. These studies show that 4 of the 5 disulphide bonds
(A to E, B to C, D to G and F to H) are conserved, with the
fifth bond between cysteine K (which is paired with L in the
7-gliadin) and M. The third inhibitor that has been disulphide-
mapped, WAI-0.53, shares 9 cysteine residues with WAI-0.28
and RBI, lacking cysteine residue E. The absence of cysteine E
is associated with the formation of a new disulphide bond
between cysteines A and H, leaving cysteine F unpaired.
These comparisons show a high degree of conservation of
disulphide bonds within and between the gliadins and 2S al-
bumins, involving cysteine residues B to C/D, C/D to G and F
to H. In addition, the y-gliadin and 2S albumins each contain
a single additional disulphide bond, between cysteines K to L
and A to E, respectively. However, only two disulphide bonds
involving the adjacent cysteine residues (C/D) are also con-
served in the inhibitors, with differences between RBI/WAI-
0.28 on the one hand and WAI-0.53 on the other. Thus, their
different disulphide bond patterns may relate to the mainte-
nance of active sites for their target enzymes.
 Kreis, M., Forde, B.G., Rahman, S., Miflin, B.J. and Shewry,
P.R. (1985) J. Mol. Biol. 183, 499-502.
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T.A. Egorov et al./FEBS Letters 396 (1996) 285 288
51 52 62 64
C D EF
G HI 1°3
8 20 37 1 17 18 29 31
A B || C D EF
45 65 73
J G H
184 191 192 204
t I i
150 151 163
",( i i
WAI - 0.28
WAI - 0.53
7 21 29 42 4354 56
I )k I
6 20 28 41 42
K F ]
Fig. 3. Comparison of the patterns of disulphide bonds formed by 2S albumins, cereal inhibitors and gliadins. Cysteine residues are labelled
A M as discussed in the text. The disulphide maps for conglutin 6, WAI-0.28, WAI-0.53, ct-gliadin and 7-gliadin are reported in [8-14].
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