Atomic force microscopy of a hybrid high‐molecular‐weight glutenin subunit from a transgenic hexaploid wheat

Abstract

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.

Direct real‐time imaging of protein adsorption onto hydrophilic and hydrophobic surfaces

“…The height of the islands, 5 nm, is consistent with measurements of the diameter of the ω‐gliadin molecule made by Thomson and coworkers17 using STM and SAXS and therefore suggests that molecules adopt a side‐on conformation to the mica surface (i.e., with the long axis parallel to the surface). It should be noted that 5 nm is greater than the diameter expected for a purely poly‐ L ‐proline II helical structure (∼1 nm); however; it is within the range expected for the diameter of a β‐spiral consisting of poly‐ L ‐proline II and β‐reverse turn (1.2–8 nm) 8. The results for ω‐gliadin adsorption onto mica are consistent with the adsorption of the related protein C hordein onto mica, which also adsorbed in parallel to the surface and with apparent preference to the edges of preadsorbed structures 6…”

“…Adsorption to the hydrophobic HOPG surface was much more rapid than when hydrophilic mica was used, indicating that protein–protein interactions were the dominant factor in controlling the adsorption behavior in this system. Recently, McIntire et al8 have used noncontact AFM to study the conformation of the high‐molecular‐weight glutenin subunits of wheat adsorbed to hydrophilic and hydrophobic surfaces.…”

Microstructure of hydrated gluten network

“…Further studies showed that proteins form fibrils at the atomic level that possibly align side by side probably due to hydrogen bond formation (Humphris, McMaster, Miles, Gilbert, Shewry & Tatham, 2000;Mackintosh, Meade, Healy, Sutton, Larsen, Squires et al, 2009;McIntire, Lew, Adalsteins, Blechl, Anderson & Kasarda, 2005). However, it is stressed that interactions of proteins with the surface of the mica and sample deposition protocols may contribute to fibril formation.…”

“…All images are reproduced by permission. Clockwise: McMaster, Miles, Kasarda, et al, 1999;McMaster, Miles, Wannerberger, et al, 1999;McIntire et al, 2005;Jiang et al, 2008;Kontogiorgos & Goff, 2006, respectively).…”

Rheological behavior of wheat gliadins in 50% (v/v) aqueous propanol

“…Conformations and molecular interactions of wheat storage proteins have been extensively investigated using various techniques such as atomic force microscopy (AFM) (McIntire et al, 2005;McMaster et al, 2000McMaster et al, ,1999Paananen et al, 2006), Raman optical activity (ROA) (Blanch et al, 2003), Fourier transform infrared (FTIR) spectroscopy (Feeney et al, 2003;Georget and Belton, 2006;Li et al, 2006;Pezolet et al, 1992), 1 H magic angle spinning nuclear magnetic resonance (MAS NMR) (Alberti et al, 2001), small angle X-ray scattering (SAXS) (Thomson et al, 1999), circular dichroism (CD) spectroscopy (Kieffer et al, 2007), and intrinsic viscosity measurement (Cole et al, 1984;Ewart, 1980;Field et al, 1987). The interaction between layers of a-gliadin has been studied by the surface force technique (Wannerberger et al, 1997).…”