Simon M. Gilbert

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

Abstract.The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an Mr of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.

Atomic Force Microscopy (AFM) Study of Interactions of HMW Subunits of Wheat Glutenin

ABSTRACT Atomic force microscopy (AFM) has been used to study the noncovalent interactions of alkylated HMW subunit 1Dx5 and a M r 58,000 peptide derived from the central repetitive domain. Both protein and peptide align side-by-side to form fibrils, the HMW subunit forming a branched network, and the peptide forming linear rods. The N- and C-terminal domains of the subunit would, therefore, appear to contain regions that interact through noncovalent interactions in the absence of disulfide bond formation. These regions may be of importance in facilitating disulfide bond formation during protein body development.

Creating a balance—the incorporation of a HMW glutenin subunit into transgenic wheat lines

Abstract Transgenic wheat lines showing over-expression of HMW glutenin subunits provide useful tools to investigate the effects of changes in the ratio of x : y subunits on functional dough properties. Titration experiments were carried out to incorporate purified subunit 1D y 10 into transgenic lines showing over-expression of subunit 1D x 5 (B73-6-1 and B73-6-4) using a reduction–oxidation process. In all cases, the incorporation of subunit 1D y 10 decreased the D x /D y ratio and resulted in a weaker dough, as shown by a shorter mixing time and an increase in resistance breakdown. Flours of three lines (the normal lines L88-31, Olympic/Gabo and the transgenic line B72-8-11b), adjusted for equivalent protein content, glutenin/gliadin ratio and HMW/LMW glutenin ratios, were analysed in order to determine the functional importance of the ratio of D x /D y subunits. A similar experiment was carried out with the line B72-8-11b, but with the amount of D y 10 incorporated being adjusted to give the same D x /D y ratio as the Olympic/Gabo line. The creation of a balanced D x /D y ratio resulted in mixing curves with a broader BWPR. These results are consistent with the hypothesis that an appropriate ratio of x : y subunits is required for dough extensibility.

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.

Extraction, separation, and purification of wheat gluten proteins and related proteins of barley, rye, and oats.

The wheat proteins, which are active in celiac disease and other glutenrelated conditions, are defined as prolamins, in that they are soluble as individual subunits in alcohol-water mixtures, such as 50% (v/v) aqueous propan-1-ol or 60-70% (v/v) aqueous ethanol. However, in wheat grain and flour, about half of these subunits are present in polymers that are not soluble in alcohol-water mixtures unless disulfide bonds between the component subunits are reduced using an agent such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). These alcohol-insoluble polymers are traditionally called glutenins and the related alcohol-soluble monomers are called gliadins; the two groups of proteins together form the major part of the gluten fraction. Gluten can be readily prepared from wheat by washing dough to remove the bulk of the starch, cell-wall material, and soluble components. It is a cohesive viscoelastic mass that contains, in addition to the gluten proteins, small amounts of other proteins, residual starch (about 25% dry wt), and lipid.

A high resolution1H magic angle spinning NMR study of a high-Mr subunit of wheat glutenin

This work describes the application of (1)H magic angle spinning (MAS) nmr to the study of hydrated 1Dx5 wheat high-M(r) subunit. 1Dx5 is a water-insoluble 88 kDa protein, associated with good baking performance, and whose structure in the solid and low-hydration states is not known. High-resolution MAS (HR-MAS) results in a threefold resolution improvement of the (1)H spectra of the hydrated wheat protein, compared to standard MAS. The spectral resolution achieved enables, for the first time, two-dimensional nmr methods to be employed for the study of hydrated 1Dx5 and the assignment of the spectrum to be carried out on the basis of total correlated spectroscopy and (13)C/(1)H correlation experiments. Considerable shifts are observed for some resonances, relative to the chemical shifts of amino acids in solution, indicating that specific interactions occur in the hydrated protein network. Two main environments are identified for glutamine residues, Q(1) and Q(2), and these were characterized in terms of possible conformation and relative dynamics, with the basis of comparison between the single 90 degrees spectrum and the Carr-Purcel-Heiboom-Gill (CPMG) spectrum. The Q(1) residues are proposed to be situated in protein segments that adopt the beta-sheet conformation and that remain relatively hindered, possibly by hydrogen bonds involving the glutamine amide groups. On the other hand, Q(2) residues are proposed to be situated in a more mobile environment, adopting a looser conformation, possibly a beta-turn conformation. Based on the proximity of the Q(2) residues with glycine residues, as viewed by the nuclear Overhauser effect spectroscopy experiment, it is proposed that the protein segments that form the more mobile (or loop) sections of the network are rich in both glutamine and glycine residues.

Molecular structures and interactions of repetitive peptides based on HMW subunit 1Dx5

A characteristic feature of high molecular weight (HMW) subunits, as with many gluten proteins, is their large repetitive central domain. This consists of tandem and interspersed repeats based on two or three consensus motifs: a nonapeptide (Gly . T yr .T yr .Pro .Thr . S er .Pro/Leu. Gln. Gln), a hexapeptide (Pro .Gly . Gln. Gly . Gln. Gln) and, in the x-type subunits only, a tripeptide (Gly.Gln.Gln). Secondary structure prediction and spectroscopic studies of purified HMW subunits and short synthetic peptides based on the consensus repeat motifs have indicated that in solution the repetitive domain forms a loose spiral consisting of regularly repeated preverse turns’”. In the solid state numerous non-covalent intermolecular interactions exist. We have used Fourier-transform infrared spectroscopy to study perfect repeat peptides with different lengths in order to understand how this affects the molecular structures.