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Structure-function relationships of soybean proteins revealed by using recombinant systems
Enzyme and Microbial Technology 30 (2002) 284 –288
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Structure-function relationships of soybean proteins revealed by using recombinant systems Shigeru Utsumi*, Nobuyuki Maruyama, Ryouhei Satoh, Motoyasu Adachi Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan
Abstract Glycinin and -conglycinin are the major storage proteins of soybean and determine the functional properties of soybean proteins. Structure-function relationships of glycinin and -conglycinin were investigated by using Escherichia coli expression systems. Examination of functional properties of various modified versions of proglycinin A1aB1b suggests that the hydrophobicity of the C-terminal region is probably important for a high emulsifying ability, that the topology of free SH residues is closely related to the heat-induced gel forming ability and that the structural factors suitable for gelation and emulsification properties are quite different. Mutual comparison of functional properties of -conglycinin constituent subunits (␣, ␣⬘ and ) and the core regions of ␣ and ␣⬘ indicate that the core regions determine thermal stability and surface hydrophobicity, that the extension regions of ␣ and ␣⬘ contribute to high solubility and emulsifying abilities and that the carbohydrate moieties inhibit the formation of heat-induced aggregates. Analyses by chimerization of glycinin and -conglycinin suggest that structure-function relationships are different between glycinin and -conglycinin. Keywords: Soybean; -conglycinin; Glycinin; Chimera; Functional properties
1. Introduction Soybean is one of the most important food protein resources. Soybean proteins are composed of two major components, glycinin and -conglycinin, which account for around 40% and 30% of total proteins, respectively [1]. Since these two proteins determine the functional properties of soybean proteins, understanding of the structure-function relationships of these two proteins at the molecular level is desired for a directed expansion of their utilization.
2. Structure-function relationships of glycinin at the molecular level Glycinin is a member of 11S globulins and has a hexameric structure [1]. Five constituent subunits are classified into two groups (Group I: A1aB1b, A1bB2, A2B1a; Group II: A3B4, A5A4B3) according to the sequence identities among them. Each subunit is synthesized as a single polypeptide precursor (preproglycinin). The signal sequence is removed cotranslationally in the endoplasmic
* Corresponding author. E-mail address: [email protected] (S. Utsumi).
reticulum (ER), and the resultant proglycinin assemble into trimers of about 8S [2]. The proglycinin trimers move from the ER into the protein storage vacuoles, where a specific posttranslational cleavage occurs [3], resulting in the mature subunits that consisit of an acidic and a basic polypeptide linked by a disulfide bond and assemble into hexamers of about 12S [2]. We had attempted to improve the functional properties of glycinin by protein engineering based on the structural characteristics and rough structure-function relationships at the subunit level [4,5]. From the analyses of the functional properties of the modified proteins, we can approch the structure-function relationships of glycinin at the molecular level. Wright [6,7] aligned the amino acid sequences to maximize the homology among the glycinin type proteins from legume and nonlegume seeds, and suggested five genetically variable regions. Each variable region was termed I to V from the N-terminus. Variable regions are rich in hydrophilic amino acids, indicating that variable regions are present on the molecular surface. (This was confirmed by our recent X-ray crystallography [8].) Therefore, the variable regions might be modified without disturbing the overall structure of the protein. The following rough structure-function relationships were revealed by analyses of the functional properties of
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S. Utsumi et al. / Enzyme and Microbial Technology 30 (2002) 284 –288
glycinin at the subunit level using soybean cultivars containing glycinins with different subunit compositions and glycinins renatured from the isolated individual subunits and reconstituted from the isolated acidic and basic polypeptides: Hydrophobicity and structural instability are related to emulsifying ability [4]; structural instability and the position and the number of SH residues are related to heat-induced gel-forming ability [5]. Various modified glycinins were designed from three viewpoints. (1) Deletion of each variable region [4]: Since each variable region has a strong hydrophilic nature, the relative hydrophobicity of the modified glycinin is enhanced. In addition, the conformational stability might be lower than that of the native glycinin, because the inherent region is deleted. Six modified glycinins lacking each variable region (⌬I, ⌬II, ⌬III, ⌬IV, ⌬V36, ⌬V8) were designed. ⌬V36 and ⌬V8 were lacking 36 and 8 residues at their C-terminus, respectively. (2) Insertion of an oligopeptide containing four methionines [4]: Since methionines have a hydrophobic nature, insertion of an oligopeptide composed of four contiguous methionines into variable regions results in enhancement of hydrophobicity. In addition, destabilization of a glycinin molecule might occur, because a hydrophobic patch is inserted into the surface hydrophilic region. Two modified glycinins (IV⫹4Met, V⫹4Met) were designed in which four methionines were inserted into the fourth and fifth variable regions. (3) Deletion of a disulfide bond [5]: Two disulfide bonds are present in a glycinin subunit [9]. In case of A1aB1b, these are Cys12-Cys45 and Cys88-Cys298 [1,8]. Disruption of a disulfide bond results in change of the number and the topology of free SH residues and disulfide bonds, and also in destabilization of a glycinin molecule. Three modified glycinins (C12G, C88S, C12GC88S) were designed. How should the abilities of the modified glycinins to form the proper conformation and their functional properties be evaluated ? Since E. coli does not contain a maturation enzyme to process proglycinin to a mature form, an E. coli expression system gives proglycinin instead of a mature form. However, proglycinin A1aB1b expressed in E. coli can selfassemble into trimers with a secondary structure similar to that of the native glycinin. In addition, the proglycinin exhibits calcium-induced precipitation, cryoprecipitation and functional properties such as emulsifying and gel forming abilities, which are inherent to native glycinin, although these are not identical to those of the native glycinin [10]. These facts indicate that the E. coli expression system can be employed for the evaluation of the formation of the correct conformation and the functional properties of the modified glycinins. The abilities of the modified glycinins to form the correct conformation were assessed by the following three criteria [11]: (1) the solubility should be comparable to that of the native glycinin; (2) there must be self-assembly into trimers; and (3) the proteins should be stable under conditions of
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high ionic strength. These three criteria were applied to all the modified proteins designed here. Consequently, ⌬I, ⌬V8, IV⫹4Met, V⫹4Met, C12G, C88S and C12GC88S were judged to form the correct conformation [4,5]. Purification of these seven modified proglycinins was attempted, and all of them were able to be purified except C12GC88S, which was easily degraded during the purification step using low ionic strength conditions [5]. Emulsifying and heat-induced gel forming abilities of the purified modified proglycinins were compared to those of the native glycinin [4,5]. All the modified proglycinins exhibited higher emulsifying activities than did the native glycinin, especially ⌬V8 and V⫹4Met, which exhibited twice the values compared to the native glycinin. These two modified proglycinins have higher hydrophobicity at the C-terminal region than the others. Therefore, the hydrophobicity of the C-terminal regions is probably important for a high emulsifying ability of glycinin [1]. All the purified modified proglycinins formed gels by boiling [4,5]. The hardness of the normal proglycinin and ⌬V8 gels was a little lower than that of the native glycinin gels. Although the hardness of the C12G gels was similar to that of the native glycinin gels at protein concentrations higher than 6%, C12G did not form gels at a concentration of 5.6%, where the native glycinin and the normal proglycinin could form gels. The gels of ⌬I, IV⫹4Met, V⫹4Met and C88S had a hardness higher than the native glycinin gels. Especially, C88S formed hard gels even at a low protein concentration (4.4%), where the native glycinin formed very soft gels. These results suggest that (1) the disulfide bond between C12 and C45 plays an important role in the initiation of the SH/S-S exchange reaction for gelation although this disulfide bond may not be essential for gelation at higher protein concentration; (2) the topology of free SH residues is closely related to the heat-induced gel forming ability; and (3) the structural factors suitable for gelation and emulsification properties are quite different [1]. 3. Structure-function relationships of – conglycinin at the molecular level
-Conglycinin is a member of 7S globulins and has a trimeric structure [1]. Three constituent subunits are identified, ␣, ␣⬘ and . The  subunit consists of only a core region which is common to all three subunits. The ␣ and ␣⬘ subunits contain extension regions at the N-terminal side of the core regions. The extension regions are rich in acidic amino acids and exhibit 57% sequence identity [12]. The core regions exhibit high absolute homologies (90, 76 and 75% between ␣ and ␣⬘, between ␣ and  and between ␣⬘ and , respectively) [12]. All subunits are N-glycosylated [1]. For the investigation of the structure-function relationships of -conglycinin, the preparation of homogeneous trimers is necessary. However, it is very difficult to obtain a
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large amount of such homogeneous trimers from soybean seeds because of molecular heterogeneity: the presence of ten molecular species having different subunit compositions [1]. E. coli expression systems easily give such homogeneous molecular species. In addition, the systems are able to give the core regions (␣ core and ␣⬘ core) of ␣ and ␣⬘ lacking the extension regions. Although -conglycinin is a glycoprotein, recombinant -conglycinins from the systems are not glycosylated. Therefore, it is possible to elucidate the contribution of the core and extension regions and the carbohydrate moieties to the functional properties of -conglycinin by comparing their functional properties with those of the native -conglycinin. CD spectra of individual recombinant proteins indicate that (1) individual recombinant proteins formed the proper secondary structures, (2) the core regions of the ␣ and ␣⬘ subunits have secondary structures similar to that of the  subunit, and (3) the extension regions of the ␣ and ␣⬘ subunits have similar secondary structures to each other [12]. Analyses using sucrose density gradient centrifugation and gel filtration indicated that all the recombinant proteins could self-assemble into trimers [12]. These results indicate that the E. coli expression system of -conglycinin can be used for the investigation of its structure-function relationships. Solubility is a prerequisite to exert functional properties such as gelation, forming and emulsification. The solubility of individual recombinant proteins was measured at high and low ionic strengths [13]. All recombinant proteins were soluble at any pH examined at ionic strength 0.5 in accordance with the native -conglycinin. On the other hand, at ionic strength 0.08 the native -conglycinin exhibited isoelectric precipitation at pH 4 – 6. The ␣ and ␣⬘ subunits also exhibited the tendency similar to that of the native -conglycinin, although the pH range causing insolubility of the native -conglycinin at ionic strength 0.08 was narrower compared with those of the ␣ and ␣⬘ subunits. The  subunits and the deletion mutants ␣ core and ␣⬘ core were insoluble at pH ⬎ 4.8 and ionic strength 0.08. These facts indicate that (1) the carbohydrate moieties of the native -conglycinin contribute to the narrower pH range for insolubility, and (2) the extension regions play an important role in the solubility of the ␣ and ␣⬘ subunits above pH 6. Structural stability is one of the important factors related to heat-induced gel forming and emulsifying abilities. Thermal stability of individual recombinant proteins was measured at pH 7.6 and ionic strength 0.5 by differential scanning microcalorimetry [12]. The results indicate that (1) the thermal stabilities of individual subunits differed from each other and the order was  (90.8°C) ⬎ ␣⬘ (82.7°C) ⬎ ␣ (78.6°C), and (2) the core regions (␣ core, 77.3°C; ␣⬘ core, 83.3°C) prescribe the thermal stabilities of the ␣ and ␣⬘ subunits [12]. The native -conglycinin gave two peaks at 79.0°C and 83.1°C, which were close to those of the ␣ and ␣⬘ subunits, and the former peak was larger than the latter, suggesting that (1) the carbohydrate moieties do not con-
tribute to the thermal stability of the native -conglycinin, and (2) the thermal stability of heterotrimers composed of two or three kinds of subunits is conferred by the subunit having the lowest denaturation temperature among the constituent subunits. Surface hydrophobicity closely relates to emulsifying and foaming abilities. Surface hydrophobicities of individual recombinant proteins were evaluated by fluorescence caused by interaction with ANS [13]. The order of the surface hydrophobicities was ␣⬘ ⬎⬎ ␣ ⬎⬎ . The surface hydrophobicities of ␣ core and ␣⬘ core were very similar to those of the ␣ and ␣⬘ subunits, respectively, indicating that the core regions prescribe the surface hydrophobicities of the ␣ and ␣⬘ subunits. The results obtained from the analyses of solubility, structural stability and hydrophobicity suggest that emulsifying ability and the formation of heat-induced soluble aggregates are different among the constituent subunits. Emulsifying abilities of the individual recombinant proteins were assessed by measuring the sizes of particles formed by homogenization and sonication of the samples with soy oil at pH 7.6 and ionic strength 0.5 [12]. The ␣ subunit exhibited the best value (4.2 m) among the three subunits, close to that of BSA, and then ␣⬘ (16.4 m) and  (52.9 m). The deletion mutants ␣ core and ␣⬘ core exhibited poorer values (33.3 m and 46.2 m) than did the ␣ and ␣⬘ subunits, but better than the  subunit. The order of ␣ core, ␣⬘ core and  corresponded to that of their thermal stabilities, but not to that of their surface hydrophobicities. These findings indicate that the extension regions and molecular stabilities of the core regions play important roles in the emulsifying abilities of -conglycinin. Heat-induced association of proteins is an important functional property and is related to their thermal stabilities and solubilities. Protein solutions heated at pH 7.6 and ionic strength 0.5 were subjected to multiangle light scattering measurements to determine the molecular masses of aggregates [13]. The ␣ and ␣⬘ subunits formed soluble aggregates having molecular masses of 2–3 million at heating temperatures ⬎ 80°C with a concomitant decrease of the intact species. The efficiency of the conversion of the ␣ subunit to soluble aggregates at 80°C was higher than that of the ␣⬘ subunit. This is probably due to the difference in their Tm (thermal denaturation midpoint temperature) values. In contrast to the ␣ and ␣⬘ subunits, the  subunit and the deletion mutants ␣ core and ␣⬘ core formed insoluble aggregates depending on their Tm values. Although the native -conglycinin formed soluble aggregates without insoluble aggregates, the amount of soluble aggregate was less than those of the ␣ and ␣⬘ subunits. These findings indicate that (1) the abilities of the ␣ and ␣⬘ subunits to form soluble aggregates are conferred by the extension regions, and (2) the carbohydrate moities inhibit the formation of aggregates of -conglycinin.
S. Utsumi et al. / Enzyme and Microbial Technology 30 (2002) 284 –288
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Fig. 1. Schematic representation of the original and chimeric proteins. (A) -conglycinin  subunit (7N-7C); (B) proglycinin A1aB1b subunit (11N-11C); (C) a chimera 7N-11C composed of the N-terminal half of  and the C-terminal half of A1aB1b; (D) a chimera 11N-7C composed of the N-terminal half of A1aB1b and the C-terminal half of ; (E) a chimera 11N-7N composed of the N-terminal half of A1aB1b and the N-terminal half of , containing a short N-terminal part of the C-terminal half of A1aB1b; (F) a chimera 7C-11C composed of the C-terminal half of  and the C-terminal half of A1aB1b, containing short C-terminal parts of the N-terminal halves of  and A1aB1b. N and C denote N- and C-termini. The numbers of the residues are from the N-terminus. 䊐, 7N; o, 7C; d, 11N; p, 11C.
4. Analyses of structure-function relationships of glycinin and -conglycinin by chimerization Although the identity in sequence between glycinin and -conglyinin is very low, partial high homologies are pointed out by many researchers, indicating that these proteins are derived from a common ancestor [6,7,14,15]. Recently, we succeeded in X-ray crystallography of proglycinin A1aB1b [8], mature glycinin A3B4 (will be described elsewhere) and -conglycinin  [16] by using E. coli expression systems and/or mutant soybean cultivars containing glycinin or -conglycinin composed of only one type of subunit. The results demonstrated that the subunits of both proteins comprise two structurally similar units (N- and C-terminal halves) in analogy with phaseolin and canavalin. These facts suggest the possibility of the creation of chimeras between glycinin and -conglycinin subunits, which might be used for detailed analyses of the structure-function relationships of glycinin and -conglycinin. We designed four chimeras (7N-11C, 11N-7C, 11N-7N and 7C-11C) (Fig. 1) based on the three dimensional structures of proglycinin A1aB1b (11N-11C) and -conglycinin  (7N-7C) (the details will be described elsewhere). All chimeras were expressed as insoluble proteins in E. coli,
although the original proteins produced in E. coli were soluble. Renaturation of the expressed proteins from the reduced-denatured state gave soluble chimeras except 7N11C. Among them, 11N-7C and 7C-11C efficiently renatured to a soluble form. The structural and functional properties of these two chimeras were examined at pH 7.6 and ionic strength 0.5 unless otherwise noted. Both 11N-7C and 7C-11C formed secondary structures rich in -strands in accordance to the original -conglycinin  and proglycinin A1aB1b. However, analyses using sucrose density gradient centrifugation and gel filtration demonstrated that they could not self-assemble into trimers: 11N-7C existed as monomers and 50% of 7C-11C was present as monomers, the remainder being randomly associated. Both chimeras did not give a peak by differential scanning microcalorimetry at a protein concentration that was sufficient in the case of the original proteins, indicating that the structures of both chimeras are not rigid. Solubilities of the original and chimeric proteins were examined as a function of ionic strength at pH 7.6. Although -conglycinin  exhibited ionic strength-dependency (insoluble at ionic strength ⱕ0.23), the chimeras were soluble at any ionic strength examined here in analogy with proglycinin A1aB1b.
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Surface hydrophobicities of the chimeric proteins measured by using ANS were much higher than those of -conglycinin  and proglycinin A1aB1b. This is probably due to the fact that the chimeric proteins could not self-assemble into trimers. Emulsifying abilities were measured at ionic strengths 0.08 and 0.5 at pH 7.6. Both chimeric proteins exhibited better emulsifying abilities than did -conglycinin  and proglycinin A1aB1b. This is probably due to their structural flexibilities and high hydrophobicities. Hydrophobicity and structural instability are important factors for the emulsifying ability of soybean proteins, but the way they contribute to these properties differs between glycinin and -conglycinin. This suggests that elucidation of the structure-function relationships of individual food proteins is necessary.
References [1] Utsumi S, Matsumura Y, Mori T. Structure-function relationships of soy proteins. In: Damodaran S, Paraf A, editors. Food proteins and their application. New York: Marcel Dekker, 1997. p. 257–291. [2] Tumer NE, Richter JD, Nielsen NC. Structural characterization of the glycinin precursors. J Biol Chem 1982;257:4016 – 8. [3] Nielsen NC. The chemistry of legume storage proteins. Philos Trans R Soc London B 1984;304:287–96. [4] Kim C-S, Kamiya S, Sato T, Utsumi S, Kito M. Improvement of nutritional value and functional properties of soybean glycinin by protein engineering. Protein Eng 1990;3:725–31. [5] Utsumi S, Gidamis AB, Kanamori J, Kang IJ, Kito M. Effects of deletion of disulfide bonds by protein engineering on the conforma-
[6] [7] [8]
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[15]
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tion and functional properties of soybean proglycinin. J Agric Food Chem 1993;41:687–91. Wright DJ. The seed globulins. In: Hudson BJF, editor. Developments in Food Proteins Vol. 5. London: Elsevier, 1987. p. 81–157. Wright DJ. The seed globulins. In: Hudson BJF, editor. Developments in Food Proteins Vol. 6. London: Elsevier, 1988. p. 119 –78. Adachi M, Takenaka Y, Gidamis AB, Mikami B, Utsumi S. Crystal structure of soybean proglycinin A1aB1b homotrimer, J Mol Biol 2001;305:291–305. Staswick PE, Hermodson MA, Nielsen NC. Identification of the cystines which link the acidic and basic components of the glycinin subunits. J Biol Chem 1984;259:13431–5. Kim C-S, Kamiya S, Kanamori J, Utsumi S, Kito M. High-level expression, purification and functional properties of soybean proglycinin from Escherichia coli. Agric Biol Chem 1990;54:1543–50. Gidamis AB, Wright P, Haque ZU, Katsube T, Kito M, Utsumi S. Modification tolerability of soybean proglycinin. Biosci Biotechnol Biochem 1995;59:1593–5. Maruyama N, Katsube T, Wada Y, Oh MH, Barba de la Rosa AP, Okuda E, Nakagawa S, Utsumi S. The roles of the N-linked glycans and extension regions of soybean -conglycinin in folding, assembly and structural features. Eur J Biochem 1998;258:854 – 62. Maruyama N, Satoh R, Wada Y, Matsumura Y, Goto H, Okuda E, Nakagawa S, Utsumi S. Structure-physicochemical function relationships of soybean -conglycinin constituent subunits. J Agric Food Chem 1999;47:5278 – 84. Gibbs PEM, Strongin KB, McPherson A. Evolution of legume seed storage proteins—a domain common to legumins and vicilins is duplicated in vicilins. Mol Biol Evol 1989;6:614 –23. Lawrence MC, Izard T, Beuchat M, Blagrove RJ, Colman PM. Structure of phaseolin at 2.2 Å resolution. J Mol Biol 1994;238:748 – 70. Maruyama N, Adachi M, Takahashi K, Yagasaki K, Kohno M, Takenaka Y, Okuda E, Nakagawa S, Mikami B, Utsumi S. Crystal structures of recombinant and native soybean -conglycinin  homotrimers. Eur J Biochem 2001;268:3595–3604.
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Structure-function relationships of soybean proteins revealed by using recombinant systems Shigeru Utsumi*, Nobuyuki Maruyama, Ryouhei Satoh, Motoyasu Adachi Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan
Abstract Glycinin and -conglycinin are the major storage proteins of soybean and determine the functional properties of soybean proteins. Structure-function relationships of glycinin and -conglycinin were investigated by using Escherichia coli expression systems. Examination of functional properties of various modified versions of proglycinin A1aB1b suggests that the hydrophobicity of the C-terminal region is probably important for a high emulsifying ability, that the topology of free SH residues is closely related to the heat-induced gel forming ability and that the structural factors suitable for gelation and emulsification properties are quite different. Mutual comparison of functional properties of -conglycinin constituent subunits (␣, ␣⬘ and ) and the core regions of ␣ and ␣⬘ indicate that the core regions determine thermal stability and surface hydrophobicity, that the extension regions of ␣ and ␣⬘ contribute to high solubility and emulsifying abilities and that the carbohydrate moieties inhibit the formation of heat-induced aggregates. Analyses by chimerization of glycinin and -conglycinin suggest that structure-function relationships are different between glycinin and -conglycinin. Keywords: Soybean; -conglycinin; Glycinin; Chimera; Functional properties
1. Introduction Soybean is one of the most important food protein resources. Soybean proteins are composed of two major components, glycinin and -conglycinin, which account for around 40% and 30% of total proteins, respectively [1]. Since these two proteins determine the functional properties of soybean proteins, understanding of the structure-function relationships of these two proteins at the molecular level is desired for a directed expansion of their utilization.
2. Structure-function relationships of glycinin at the molecular level Glycinin is a member of 11S globulins and has a hexameric structure [1]. Five constituent subunits are classified into two groups (Group I: A1aB1b, A1bB2, A2B1a; Group II: A3B4, A5A4B3) according to the sequence identities among them. Each subunit is synthesized as a single polypeptide precursor (preproglycinin). The signal sequence is removed cotranslationally in the endoplasmic
* Corresponding author. E-mail address: [email protected] (S. Utsumi).
reticulum (ER), and the resultant proglycinin assemble into trimers of about 8S [2]. The proglycinin trimers move from the ER into the protein storage vacuoles, where a specific posttranslational cleavage occurs [3], resulting in the mature subunits that consisit of an acidic and a basic polypeptide linked by a disulfide bond and assemble into hexamers of about 12S [2]. We had attempted to improve the functional properties of glycinin by protein engineering based on the structural characteristics and rough structure-function relationships at the subunit level [4,5]. From the analyses of the functional properties of the modified proteins, we can approch the structure-function relationships of glycinin at the molecular level. Wright [6,7] aligned the amino acid sequences to maximize the homology among the glycinin type proteins from legume and nonlegume seeds, and suggested five genetically variable regions. Each variable region was termed I to V from the N-terminus. Variable regions are rich in hydrophilic amino acids, indicating that variable regions are present on the molecular surface. (This was confirmed by our recent X-ray crystallography [8].) Therefore, the variable regions might be modified without disturbing the overall structure of the protein. The following rough structure-function relationships were revealed by analyses of the functional properties of
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 5 0 7 - 5
S. Utsumi et al. / Enzyme and Microbial Technology 30 (2002) 284 –288
glycinin at the subunit level using soybean cultivars containing glycinins with different subunit compositions and glycinins renatured from the isolated individual subunits and reconstituted from the isolated acidic and basic polypeptides: Hydrophobicity and structural instability are related to emulsifying ability [4]; structural instability and the position and the number of SH residues are related to heat-induced gel-forming ability [5]. Various modified glycinins were designed from three viewpoints. (1) Deletion of each variable region [4]: Since each variable region has a strong hydrophilic nature, the relative hydrophobicity of the modified glycinin is enhanced. In addition, the conformational stability might be lower than that of the native glycinin, because the inherent region is deleted. Six modified glycinins lacking each variable region (⌬I, ⌬II, ⌬III, ⌬IV, ⌬V36, ⌬V8) were designed. ⌬V36 and ⌬V8 were lacking 36 and 8 residues at their C-terminus, respectively. (2) Insertion of an oligopeptide containing four methionines [4]: Since methionines have a hydrophobic nature, insertion of an oligopeptide composed of four contiguous methionines into variable regions results in enhancement of hydrophobicity. In addition, destabilization of a glycinin molecule might occur, because a hydrophobic patch is inserted into the surface hydrophilic region. Two modified glycinins (IV⫹4Met, V⫹4Met) were designed in which four methionines were inserted into the fourth and fifth variable regions. (3) Deletion of a disulfide bond [5]: Two disulfide bonds are present in a glycinin subunit [9]. In case of A1aB1b, these are Cys12-Cys45 and Cys88-Cys298 [1,8]. Disruption of a disulfide bond results in change of the number and the topology of free SH residues and disulfide bonds, and also in destabilization of a glycinin molecule. Three modified glycinins (C12G, C88S, C12GC88S) were designed. How should the abilities of the modified glycinins to form the proper conformation and their functional properties be evaluated ? Since E. coli does not contain a maturation enzyme to process proglycinin to a mature form, an E. coli expression system gives proglycinin instead of a mature form. However, proglycinin A1aB1b expressed in E. coli can selfassemble into trimers with a secondary structure similar to that of the native glycinin. In addition, the proglycinin exhibits calcium-induced precipitation, cryoprecipitation and functional properties such as emulsifying and gel forming abilities, which are inherent to native glycinin, although these are not identical to those of the native glycinin [10]. These facts indicate that the E. coli expression system can be employed for the evaluation of the formation of the correct conformation and the functional properties of the modified glycinins. The abilities of the modified glycinins to form the correct conformation were assessed by the following three criteria [11]: (1) the solubility should be comparable to that of the native glycinin; (2) there must be self-assembly into trimers; and (3) the proteins should be stable under conditions of
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high ionic strength. These three criteria were applied to all the modified proteins designed here. Consequently, ⌬I, ⌬V8, IV⫹4Met, V⫹4Met, C12G, C88S and C12GC88S were judged to form the correct conformation [4,5]. Purification of these seven modified proglycinins was attempted, and all of them were able to be purified except C12GC88S, which was easily degraded during the purification step using low ionic strength conditions [5]. Emulsifying and heat-induced gel forming abilities of the purified modified proglycinins were compared to those of the native glycinin [4,5]. All the modified proglycinins exhibited higher emulsifying activities than did the native glycinin, especially ⌬V8 and V⫹4Met, which exhibited twice the values compared to the native glycinin. These two modified proglycinins have higher hydrophobicity at the C-terminal region than the others. Therefore, the hydrophobicity of the C-terminal regions is probably important for a high emulsifying ability of glycinin [1]. All the purified modified proglycinins formed gels by boiling [4,5]. The hardness of the normal proglycinin and ⌬V8 gels was a little lower than that of the native glycinin gels. Although the hardness of the C12G gels was similar to that of the native glycinin gels at protein concentrations higher than 6%, C12G did not form gels at a concentration of 5.6%, where the native glycinin and the normal proglycinin could form gels. The gels of ⌬I, IV⫹4Met, V⫹4Met and C88S had a hardness higher than the native glycinin gels. Especially, C88S formed hard gels even at a low protein concentration (4.4%), where the native glycinin formed very soft gels. These results suggest that (1) the disulfide bond between C12 and C45 plays an important role in the initiation of the SH/S-S exchange reaction for gelation although this disulfide bond may not be essential for gelation at higher protein concentration; (2) the topology of free SH residues is closely related to the heat-induced gel forming ability; and (3) the structural factors suitable for gelation and emulsification properties are quite different [1]. 3. Structure-function relationships of – conglycinin at the molecular level
-Conglycinin is a member of 7S globulins and has a trimeric structure [1]. Three constituent subunits are identified, ␣, ␣⬘ and . The  subunit consists of only a core region which is common to all three subunits. The ␣ and ␣⬘ subunits contain extension regions at the N-terminal side of the core regions. The extension regions are rich in acidic amino acids and exhibit 57% sequence identity [12]. The core regions exhibit high absolute homologies (90, 76 and 75% between ␣ and ␣⬘, between ␣ and  and between ␣⬘ and , respectively) [12]. All subunits are N-glycosylated [1]. For the investigation of the structure-function relationships of -conglycinin, the preparation of homogeneous trimers is necessary. However, it is very difficult to obtain a
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large amount of such homogeneous trimers from soybean seeds because of molecular heterogeneity: the presence of ten molecular species having different subunit compositions [1]. E. coli expression systems easily give such homogeneous molecular species. In addition, the systems are able to give the core regions (␣ core and ␣⬘ core) of ␣ and ␣⬘ lacking the extension regions. Although -conglycinin is a glycoprotein, recombinant -conglycinins from the systems are not glycosylated. Therefore, it is possible to elucidate the contribution of the core and extension regions and the carbohydrate moieties to the functional properties of -conglycinin by comparing their functional properties with those of the native -conglycinin. CD spectra of individual recombinant proteins indicate that (1) individual recombinant proteins formed the proper secondary structures, (2) the core regions of the ␣ and ␣⬘ subunits have secondary structures similar to that of the  subunit, and (3) the extension regions of the ␣ and ␣⬘ subunits have similar secondary structures to each other [12]. Analyses using sucrose density gradient centrifugation and gel filtration indicated that all the recombinant proteins could self-assemble into trimers [12]. These results indicate that the E. coli expression system of -conglycinin can be used for the investigation of its structure-function relationships. Solubility is a prerequisite to exert functional properties such as gelation, forming and emulsification. The solubility of individual recombinant proteins was measured at high and low ionic strengths [13]. All recombinant proteins were soluble at any pH examined at ionic strength 0.5 in accordance with the native -conglycinin. On the other hand, at ionic strength 0.08 the native -conglycinin exhibited isoelectric precipitation at pH 4 – 6. The ␣ and ␣⬘ subunits also exhibited the tendency similar to that of the native -conglycinin, although the pH range causing insolubility of the native -conglycinin at ionic strength 0.08 was narrower compared with those of the ␣ and ␣⬘ subunits. The  subunits and the deletion mutants ␣ core and ␣⬘ core were insoluble at pH ⬎ 4.8 and ionic strength 0.08. These facts indicate that (1) the carbohydrate moieties of the native -conglycinin contribute to the narrower pH range for insolubility, and (2) the extension regions play an important role in the solubility of the ␣ and ␣⬘ subunits above pH 6. Structural stability is one of the important factors related to heat-induced gel forming and emulsifying abilities. Thermal stability of individual recombinant proteins was measured at pH 7.6 and ionic strength 0.5 by differential scanning microcalorimetry [12]. The results indicate that (1) the thermal stabilities of individual subunits differed from each other and the order was  (90.8°C) ⬎ ␣⬘ (82.7°C) ⬎ ␣ (78.6°C), and (2) the core regions (␣ core, 77.3°C; ␣⬘ core, 83.3°C) prescribe the thermal stabilities of the ␣ and ␣⬘ subunits [12]. The native -conglycinin gave two peaks at 79.0°C and 83.1°C, which were close to those of the ␣ and ␣⬘ subunits, and the former peak was larger than the latter, suggesting that (1) the carbohydrate moieties do not con-
tribute to the thermal stability of the native -conglycinin, and (2) the thermal stability of heterotrimers composed of two or three kinds of subunits is conferred by the subunit having the lowest denaturation temperature among the constituent subunits. Surface hydrophobicity closely relates to emulsifying and foaming abilities. Surface hydrophobicities of individual recombinant proteins were evaluated by fluorescence caused by interaction with ANS [13]. The order of the surface hydrophobicities was ␣⬘ ⬎⬎ ␣ ⬎⬎ . The surface hydrophobicities of ␣ core and ␣⬘ core were very similar to those of the ␣ and ␣⬘ subunits, respectively, indicating that the core regions prescribe the surface hydrophobicities of the ␣ and ␣⬘ subunits. The results obtained from the analyses of solubility, structural stability and hydrophobicity suggest that emulsifying ability and the formation of heat-induced soluble aggregates are different among the constituent subunits. Emulsifying abilities of the individual recombinant proteins were assessed by measuring the sizes of particles formed by homogenization and sonication of the samples with soy oil at pH 7.6 and ionic strength 0.5 [12]. The ␣ subunit exhibited the best value (4.2 m) among the three subunits, close to that of BSA, and then ␣⬘ (16.4 m) and  (52.9 m). The deletion mutants ␣ core and ␣⬘ core exhibited poorer values (33.3 m and 46.2 m) than did the ␣ and ␣⬘ subunits, but better than the  subunit. The order of ␣ core, ␣⬘ core and  corresponded to that of their thermal stabilities, but not to that of their surface hydrophobicities. These findings indicate that the extension regions and molecular stabilities of the core regions play important roles in the emulsifying abilities of -conglycinin. Heat-induced association of proteins is an important functional property and is related to their thermal stabilities and solubilities. Protein solutions heated at pH 7.6 and ionic strength 0.5 were subjected to multiangle light scattering measurements to determine the molecular masses of aggregates [13]. The ␣ and ␣⬘ subunits formed soluble aggregates having molecular masses of 2–3 million at heating temperatures ⬎ 80°C with a concomitant decrease of the intact species. The efficiency of the conversion of the ␣ subunit to soluble aggregates at 80°C was higher than that of the ␣⬘ subunit. This is probably due to the difference in their Tm (thermal denaturation midpoint temperature) values. In contrast to the ␣ and ␣⬘ subunits, the  subunit and the deletion mutants ␣ core and ␣⬘ core formed insoluble aggregates depending on their Tm values. Although the native -conglycinin formed soluble aggregates without insoluble aggregates, the amount of soluble aggregate was less than those of the ␣ and ␣⬘ subunits. These findings indicate that (1) the abilities of the ␣ and ␣⬘ subunits to form soluble aggregates are conferred by the extension regions, and (2) the carbohydrate moities inhibit the formation of aggregates of -conglycinin.
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Fig. 1. Schematic representation of the original and chimeric proteins. (A) -conglycinin  subunit (7N-7C); (B) proglycinin A1aB1b subunit (11N-11C); (C) a chimera 7N-11C composed of the N-terminal half of  and the C-terminal half of A1aB1b; (D) a chimera 11N-7C composed of the N-terminal half of A1aB1b and the C-terminal half of ; (E) a chimera 11N-7N composed of the N-terminal half of A1aB1b and the N-terminal half of , containing a short N-terminal part of the C-terminal half of A1aB1b; (F) a chimera 7C-11C composed of the C-terminal half of  and the C-terminal half of A1aB1b, containing short C-terminal parts of the N-terminal halves of  and A1aB1b. N and C denote N- and C-termini. The numbers of the residues are from the N-terminus. 䊐, 7N; o, 7C; d, 11N; p, 11C.
4. Analyses of structure-function relationships of glycinin and -conglycinin by chimerization Although the identity in sequence between glycinin and -conglyinin is very low, partial high homologies are pointed out by many researchers, indicating that these proteins are derived from a common ancestor [6,7,14,15]. Recently, we succeeded in X-ray crystallography of proglycinin A1aB1b [8], mature glycinin A3B4 (will be described elsewhere) and -conglycinin  [16] by using E. coli expression systems and/or mutant soybean cultivars containing glycinin or -conglycinin composed of only one type of subunit. The results demonstrated that the subunits of both proteins comprise two structurally similar units (N- and C-terminal halves) in analogy with phaseolin and canavalin. These facts suggest the possibility of the creation of chimeras between glycinin and -conglycinin subunits, which might be used for detailed analyses of the structure-function relationships of glycinin and -conglycinin. We designed four chimeras (7N-11C, 11N-7C, 11N-7N and 7C-11C) (Fig. 1) based on the three dimensional structures of proglycinin A1aB1b (11N-11C) and -conglycinin  (7N-7C) (the details will be described elsewhere). All chimeras were expressed as insoluble proteins in E. coli,
although the original proteins produced in E. coli were soluble. Renaturation of the expressed proteins from the reduced-denatured state gave soluble chimeras except 7N11C. Among them, 11N-7C and 7C-11C efficiently renatured to a soluble form. The structural and functional properties of these two chimeras were examined at pH 7.6 and ionic strength 0.5 unless otherwise noted. Both 11N-7C and 7C-11C formed secondary structures rich in -strands in accordance to the original -conglycinin  and proglycinin A1aB1b. However, analyses using sucrose density gradient centrifugation and gel filtration demonstrated that they could not self-assemble into trimers: 11N-7C existed as monomers and 50% of 7C-11C was present as monomers, the remainder being randomly associated. Both chimeras did not give a peak by differential scanning microcalorimetry at a protein concentration that was sufficient in the case of the original proteins, indicating that the structures of both chimeras are not rigid. Solubilities of the original and chimeric proteins were examined as a function of ionic strength at pH 7.6. Although -conglycinin  exhibited ionic strength-dependency (insoluble at ionic strength ⱕ0.23), the chimeras were soluble at any ionic strength examined here in analogy with proglycinin A1aB1b.
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Surface hydrophobicities of the chimeric proteins measured by using ANS were much higher than those of -conglycinin  and proglycinin A1aB1b. This is probably due to the fact that the chimeric proteins could not self-assemble into trimers. Emulsifying abilities were measured at ionic strengths 0.08 and 0.5 at pH 7.6. Both chimeric proteins exhibited better emulsifying abilities than did -conglycinin  and proglycinin A1aB1b. This is probably due to their structural flexibilities and high hydrophobicities. Hydrophobicity and structural instability are important factors for the emulsifying ability of soybean proteins, but the way they contribute to these properties differs between glycinin and -conglycinin. This suggests that elucidation of the structure-function relationships of individual food proteins is necessary.
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