Atomic force microscopy of a hybrid high-molecular-weight glutenin subunit from a transgenic hexaploid wheat.
The high-molecular-weight glutenin subunits (HMW-GS) of wheat gluten in their native form are incorporated into an intermolecularly disulfide-linked, polymeric system that gives rise to the elasticity of wheat flour doughs. These protein subunits range in molecular weight from about 70 K-90 K and are made up of small N-terminal and C-terminal domains and a large central domain that consists of repeating sequences rich in glutamine, proline, and glycine. The cysteines involved in forming intra- and intermolecular disulfide bonds are found in, or close to, the N- and C-terminal domains. A model has been proposed in which the repeating sequence domain of the HMW-GS forms a rod-like beta-spiral with length near 50 nm and diameter near 2 nm. We have sought to examine this model by using noncontact atomic force microscopy (NCAFM) to image a hybrid HMW-GS in which the N-terminal domain of subunit Dy10 has replaced the N-terminal domain of subunit Dx5. This hybrid subunit, coded by a transgene overexpressed in transgenic wheat, has the unusual characteristic of forming, in vivo, not only polymeric forms, but also a monomer in which a single disulfide bond links the C-terminal domain to the N-terminal domain, replacing the two intermolecular disulfide bonds normally formed by the corresponding cysteine side chains. No such monomeric subunits have been observed in normal wheat lines, only polymeric forms. NCAFM of the native, unreduced 93 K monomer showed fibrils of varying lengths but a length of about 110 nm was particularly noticeable whereas the reduced form showed rod-like structures with a length of about 300 nm or greater. The 110 nm fibrils may represent the length of the disulfide-linked monomer, in which case they would not be in accord with the beta-spiral model, but would favor a more extended conformation for the polypeptide chain, possibly polyproline II.
McIntire
Theresa M
TM
Department of Chemistry, University of California, Irvine, Irvine, CA 92697-2025, USA.
Lew
Ellen J L
EJ
Adalsteins
A Elva
AE
Blechl
Ann
A
Anderson
Olin D
OD
Brant
David A
DA
Kasarda
Donald D
DD
D004220:Disulfides / Q000737:chemistry; D005983:Glutens / Q000031:analogs & derivatives* / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008970:Molecular Weight; D030821:Plants, Genetically Modified; D017434:Protein Structure, Tertiary; D014908:Triticum / Q000737:chemistry*
D004220:Disulfides; D005983:Glutens; C037302:glutenin