Pconglycinin

We analyzed the structural features of the native homotrimers (a3, a'3, and P3) of the individual subunits of P-conglycinin prepared from seeds of mutant cultivars containing P-conglycinin with limited subunit compositions, the recombinant homotrimers (a3, a'3, P3, acore3, and a'core3) of individual subunits, and the acore and a'core of P-conglycinin by means of E. coli expression system (35-37).

On a sucrose density gradient centrifugation, three recombinant homotrimers sedi-mented at similar positions. However, the recombinant a3 and a'3 eluted faster than did the recombinant P3 on a gel filtration chromatography, similarly to the native glycinin hex-amer. These indicate that the extension regions largely contribute to the dimensions of a3 and a'3. Therefore, the structural models indicated in Figure 2.8 can be proposed. The order of thermal stability of the recombinant and native homotrimers was a3 < a'3 < P3, and P3 was the most stable among the three subunits. On the other hand, the acore3 and a'core3 exhibited similar thermal stability to those of a3 and a'3, respectively. These results indicate that the extension regions and the glycans do not affect the thermal stability, and that the core regions determine the thermal stability of the individual subunits of P-conglycinin. Further, an analysis of the thermal stability of the heterotrimers of P-conglycinin indicated that thermal stabilities of most heterotrimers are determined by that of the constituent subunit which has the lower thermal stability (38). On the other hand, the order of surface hydrophobicity was a3 > a'3 $ P3, and dependent on that of the core regions. Surface hydrophobicities of the heterotrimers were the arithmetic mean of those of constituent sub-units. The extension regions and the glycans largely affect solubility under lower ionic strength, but not under higher ionic strength. The solubility of the trimer containing even one a or a' subunit is much higher than that of the P3, acore3 and a'core3, and close to that of the a3 and a'3, indicating that the extension regions contribute largely to the solubility.

Because the thermal stability of the core region determines that of the subunit, we tried to analyze the structural factors related to thermal stability by comparing the three dimensional structure of the a'core3 with that of P3 in detail. Thus, five structural factors that can account for the result that P3 is more thermostable than a'core3 were elucidated (39).

First, protein packs tightly but some cavities are often observed inside the molecule, resulting in a decrease in its thermal stability (40). We calculated the total cavity volume

Figure 2.8 Structure models of soybean P-conglycinin. (a) P3, (b) a3 or a3. The core and the extension regions are shown in ball and stick, respectively.

of the a'core3 (172 A3) to be larger than that of P3 (79 A3), indicating that P3 is more tightly packed than a'core3. The main cavities are located in the P-barrels of the N- and C-terminal modules. The cavities of the C-terminal P-barrels of P3 and a'core3 exhibit a prominent difference between them. Twelve residues among 15 residues lining the cavity were common between the P and a'core, and three residues were different. In other words, Phe233, Leu262, and Phe325 of P were substituted with Leu376, Phe405, and Val468 of a'core, respectively. Because of the compensating substitutions of Leu376 and Phe405 in the a'core to Phe and Leu in P, respectively, a substitution of Val468 in the a'core to Phe in P contributes mainly to the difference in the cavity volume of the C-terminal P-barrel.

Second, because long ion pair networks have been found in several enzymes from hyperthermophilic bacterium (41,42), they are thought to be a common mechanism to stabilize the protein of hyperthermophiles (43). Sequence alignment of P and a'core indicates that some of the charged residues in P are substituted with noncharged residues in the a'core. These residues exist in the a-helix domain of the C-terminal module, and form a cluster at the intermonomer interface. The length of an ion pair network of P3 was longer than that of the a'core3, because E353, R359, E362, R363, R379, K383, and R385 of P were replaced with S496, S502, P505, S506, N522, S526, and S528 of the a'core, respectively.

Third, the hydration of nonpolar groups apparently destabilizes proteins (44). Experimental analysis of surface hydrophobicity using a hydrophobic column demonstrated that the molecular surface of a'core3 was more hydrophobic than that of P3. Further, a comparison of three dimensional structures between P3 and a'core3 indicated that the ratio of hydrophobic atoms (carbon and sulfur) to all atoms of solvent accessible surface of a'core3 was higher than that of P3.

Fourth, the number of proline residues, which stabilize the protein structure by decreasing the entropy of denatured structure, is 57 in the a core3 compared to 63 in P3. According to Matthews et al. (45), this difference contributes 4.8 kcal/mol to the AG. Therefore, the difference in the number of proline residues may affect the thermal stability.

Fifth, shorter loops are one of stabilizing factors in thermophilic bacteria (46). The loop between helix 3 and strand J' is five residues shorter in P than that in a core. The electron densities of this loop region were broken in all monomers of the a core3 and two of P3, but appeared in one monomer of P3, giving the clearest density map among the three monomers. Because the resolution of P3 is lower, this may suggest relatively rigid conformation of this loop region in the P subunit. The length of the loop is also considered one of the important factors related to the structural stability of the cupin superfamily to which P-conglycinin belongs.

As described, we investigated possible factors for the difference in the thermal stability between the a 'core3 and P3 through the comparison of three dimensional structures. More hydrogen bonds were observed in each module of a'core3, which suggests more stable packing of a 'core3, and is not consistent with the experimental data. This difference should be more than compensated by the accumulation of the effects of many factors which account for less thermal stability of the a'core3, such as larger cavity volumes, lack of short ion pair networks, higher surface hydrophobicity, lower number of proline residues, and a longer loop. We think that P3 would be stabilized more than the a'core3 by the sum of the contributions of each of these factors.

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