Synthesis and assembly of soybean beta-conglycinin in vitro.
The construction of SP6-derived expression plasmids that encode normal and modified beta-conglycinin subunits is described. With the exception of an additional methionine at their NH2-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids corresponded to mature alpha and beta subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH2-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal alpha subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified beta-conglycinin subunits. The results also show that structural changes at the NH2-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.
Lelievre
J M
JM
USDA Agricultural Research Service, Purdue University, West Lafayette, IN 47907.
Dickinson
C D
CD
Dickinson
L A
LA
Nielsen
N C
NC
D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004586:Electrophoresis; D005916:Globulins / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D014674:Plant Proteins, Dietary / Q000378:metabolism*; D055314:Seed Storage Proteins; D030262:Soybean Proteins; D013025:Glycine max / Q000378:metabolism*; D013329:Structure-Activity Relationship
D052179:Antigens, Plant; D005916:Globulins; D046911:Macromolecular Substances; D014674:Plant Proteins, Dietary; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max