Received: 22 February 2022 | Revised: 11 May 2022 | Accepted: 25 May 2022

DOI: 10.1002/cche.10572

REVIEW

Chemistry of wheat gluten proteins: Qualitative

composition

Herbert Wieser1 | Peter Koehler2 | Katharina A. Scherf3

1
Hamburg School of Food Science,
Institute of Food Chemistry, University of Abstract
Hamburg, Hamburg, Germany Background and Objectives: Wheat gluten proteins make up one of the
2
Biotask AG, Esslingen, Germany most complex protein aggregates in nature. Their qualitative and quantitative
3
Department of Bioactive and Functional
composition is determined by genetic and environmental factors as well as
Food Chemistry, Institute of Applied
Biosciences, Karlsruhe Institute of technological processes.
Technology (KIT), Karlsruhe, Germany Findings: Gluten proteins comprise ω5‐, ω1,2‐, α‐, and γ‐gliadins as well as
high‐molecular‐weight glutenin subunits (HMW‐GS) and low‐molecular‐
Correspondence
Katharina A. Scherf, Karlsruhe Institute weight (LMW) GS. About 50% of gluten proteins are monomeric gliadins
of Technology (KIT), Adenauerring 20 a, with MWs from 28,000 to 55,000, while about 15% are present as disulfide‐
76131 Karlsruhe, Germany.
Email: [email protected] and
linked oligomeric proteins with MWs between 70,000 and 700,000, called
bioactivefc.iab.kit.edu HMW‐gliadins. The remaining 35% are disulfide‐linked polymeric glutenins
with MWs from 700,000 to more than 10 million. Intrachain disulfide bonds,
Funding information
present in all types except ω‐gliadins, stabilize the three‐dimensional
Bundesministerium für Ernährung und
Landwirtschaft, Grant/Award Number: structure, while interchain disulfide bonds, mainly linking HMW‐GS and
2818404B18 LMW‐GS, generate oligomers and polymers.
Conclusions: In this review, we provide an updated and detailed insight into
the chemistry of wheat gluten proteins with a focus on the qualitative
composition.
Significance and Novelty: An enhanced understanding of gluten protein
structure and how it is affected will be essential to select and breed more
resilient wheat varieties with favorable processing properties to help ensure
nutrition and food security worldwide.

KEYWORDS
amino acid sequences, bread making, disulfide bonds, gliadin, glutenin, polymer

1 | INTRODUCTION and are essential pillars for food security worldwide.

Today, wheat is grown on about 220 million hectares
Wheat is among the top three of the world's most of arable land, particularly in temperate regions,
important crops cultivated by mankind. Wheat‐based and yields around 770 million tons of grains per
products such as bread, other baked goods, pasta, and year (FAOSTAT, 2022). Common wheat (Triticum
noodles have been staple foods for thousands of years aestivum L.), also known as bread wheat, is the most
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2022 The Authors. Cereal Chemistry published by Wiley Periodicals LLC on behalf of Cereals & Grains Association.

Cereal Chemistry. 2023;100:23–35. wileyonlinelibrary.com/journal/cche | 23

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24 | WIESER ET AL.

widespread wheat species and represents around 95% 2.1 | Protein fractions
of cultivated wheat. Most of the remaining 5% are
durum wheat (Triticum durum L.), well‐known as Wheat grain proteins have been traditionally classified
pasta wheat. The other wheat species, einkorn into four so‐called Osborne fractions according to
(Triticum monococcum L.), emmer (Triticum dicoccum different solubility: albumins, globulins, gliadins, and
(Schrank) Schübler), and spelt (Triticum spelta L.) glutenins (Osborne, 1907), along with residual proteins
only play a minor role in terms of utilization and are (Figure 1). Albumins are soluble in water and dilute salt
processed into specialty products. The success of solutions, while globulins are soluble in dilute salt
wheat is largely due to the special chemical and solutions, but not in water. The albumin/globulin
physical properties of its gluten proteins that make up fraction mainly contains regulatory, metabolic, and
the essence of wheat uniqueness. They correspond to protective proteins such as enzymes and enzyme inhibi-
the storage proteins of wheat and represent around tors. Gliadins are soluble in aqueous alcohols, for
70%–80% of total grain proteins (P. Shewry, 2019; example, 60% ethanol or 50% propanol, but insoluble in
Wieser et al., 2020). water and salt solutions. They can be subdivided into
Gluten proteins make up one of the most complex monomeric proteins and oligomeric proteins, so‐called
protein aggregates in nature. Sophisticated separation high‐molecular‐weight (HMW)‐gliadins (Schmid et al.,
procedures and analytical methods now allow investiga- 2016). Native polymeric glutenins are insoluble in the
tions of the structure of single proteins and their intra‐ and three solvents mentioned, but they can be solubilized as
intermolecular linkages. Both genetic and environmental monomeric glutenin subunits (GS) after reduction of
factors as well as technological processes determine the disulfide (SS) bonds, in, for example, 50% propanol
qualitative and quantitative composition of gluten containing a reducing agent such as dithiothreitol. A
proteins. In this review, we provide an updated and small portion of proteins including membrane proteins
detailed insight into the chemistry of wheat gluten and lipoproteins does not belong to any of the Osborne
proteins with a focus on the qualitative composition. fractions. Together with starch, they remain in the
Different factors determining the quantitative composition insoluble residue, after the Osborne fractions have been
of gluten are available in the associated review by Wieser extracted (Wieser, 2007).
et al. (2022).

2 | CLASSIFICATION OF WHEAT
PROTEINS

Proteomic separation of wheat grain proteins by two‐

dimensional gel electrophoresis (2D‐GE) resolved up to
1300 proteins (Skylas et al., 2000). Many of these are
closely related due to protein polymorphism, the
presence of multigene families, and posttranslational
modifications. Corresponding to the number of sub-
genomes, hexaploid common wheat and spelt (genome
AABBDD, 42 chromosomes) contain the highest
number of proteins, followed by tetraploid durum
wheat and emmer (genome AABB, 28 chromosomes).
Diploid einkorn (genome AA, 14 chromosomes) has
the lowest number of proteins. For example, 476
protein spots were detected in a total protein extract
from common wheat flour by 2D‐GE. Of the 233 F I G U R E 1 Classification of wheat proteins into different
proteins subsequently identified by liquid chromatog- fractions and types. The percentages are typical values for each
raphy with tandem mass spectrometry (LC‐MS/MS), gluten protein type relative to the total amount of gluten
122 were gluten proteins (Dupont et al., 2011). 2D‐GE determined by modified Osborne fractionation and reversed‐phase
analysis of 10 durum wheat cultivars revealed 51 to 75 high‐performance liquid chromatography as reported by Lexhaller
gluten proteins (de Angelis et al., 2008), whereas et al. (2017). GS, glutenin subunits; HMW, high molecular weight;
einkorn contained about 2030 gluten proteins (Alvarez LMW, low molecular weight; m, monomeric; MMW, medium
et al., 2006). molecular weight; MW, molecular weight; p, polymeric.
 19433638, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cche.10572, Wiley Online Library on [20/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
WIESER ET AL. | 25

Gliadins and glutenins are the storage proteins of the

grain, also known as gluten proteins. Exclusively located
in the starchy endosperm, they represent around 70%
to 80% of total grain protein. Their synthesis starts
around 10 days after the flowering of the plants and ends
around 20 days later. They are deposited in discrete
protein bodies within the starchy endosperm cells. After
coalescence of the protein bodies during grain matura-
tion, gliadins and glutenins form a continuous gluten
network, in which the starch granules are embedded
(Tosi, 2012). Analysis of wheat pearling fractions
demonstrated that there is a quantitative and qualitative
protein gradient in the starchy endosperm, with HMW‐
GS mainly located in the central endosperm (He
et al., 2013). The main biological function of gluten
proteins is to supply the wheat seedling with nitrogen,
amino acids, and energy during germination. From a
processing point of view, gluten proteins are what make
wheat unique in bread making and multiple other
applications. When water is added to wheat flour,
gliadins and glutenins develop a cohesive viscoelastic F I G U R E 2 Sodium dodecyl sulfate‐polyacrylamide gel
gluten network during mixing that retains the gas electrophoresis of wheat flour proteins illustrating the molecular
generated during dough fermentation and leads to the weight distribution of the proteins under reducing conditions. 1,
formation of a leavened, fluffy bread crumb (Delcour variety Tuareg; 2, wheat flour blend of the varieties Soissons,
Bezostaya‐1, Glenlea, Hereward, and Mv Magvas; 3, variety
et al., 2012).
Soissons; 4, variety Glenlea; 5, variety Hereward. GS, glutenin
subunits; HMW, high molecular weight; LMW, low molecular
weight.
2.2 | Gluten protein types

Gluten proteins are classified into six different types into x‐type and y‐type with about 800 and 600 amino
according to similarities and differences in the amino acid residues equivalent to MWs of 83,000 to 88,000 and
acid sequences, respectively (Figure 1). Gliadins are 67,000 to 74,000, respectively (Juhász & Gianibelli, 2006;
subdivided into α‐, γ‐, ω1,2‐, and ω5‐gliadins according Metakovsky et al., 2006; P. R. Shewry et al., 2006).
to different mobility upon acid PAGE, whereas glute- Gliadin types are mainly encoded on the short arms of
nins consist of HMW‐GS and low‐molecular‐weight chromosomes 1A, 1B, 1D, 6A, and 6B, but some γ‐gliadins
(LMW)‐GS based on the molecular weights (MWs) are also on the long arms of chromosomes 3B and 3D.
observed by sodium dodecyl sulfate‐polyacrylamide gel LMW‐GS genes are located on the short arms of
electrophoresis (SDS‐PAGE) (Figure 2). α‐Gliadins, chromosomes 1A, 1B, and 1D, whereas HMW‐GS genes
γ‐gliadins, and LMW‐GS belong to the LMW‐group of are on the long arms of the same group 1 chromosome
gluten protein types with a length of 260 to 330 amino (Juhász et al., 2018). Due to their particular importance for
acid residues, which corresponds to MWs from 28,000 to dough and bread quality, single HMW‐GS are named after
35,000. The medium‐molecular‐weight (MMW) group their gene loci (1A, 1B, or 1D chromosome), their size
comprises ω1,2‐gliadins with about 370 amino acid (x‐type or y‐type), and their mobility upon SDS‐PAGE
residues and MWs of 39,000 to 44,000 as well as (numbered 1–12) in many cases. HMW‐GS 1Dx5, for
ω5‐gliadins with about 420 amino acid residues and example, is encoded on chromosome 1D, belongs to the
MWs of 49,000 to 55,000. A small portion of ω‐type x‐type, and appears at position 5 on SDS‐PAGE. The
gliadins, called glutenin‐bound gliadins (ωb‐gliadins) is criterion of SDS‐PAGE mobility always needs to be verified
modified by substitution of a single amino acid residue for each specific gel and buffer combination, because
for a cysteine residue that is linked to other gluten mobility can be altered. The elution order of HMW‐GS in a
proteins by an SS bond. This is the reason why ωb‐ Bis‐Tris SDS‐PAGE system with a neutral running buffer
gliadins appear in the glutenin fraction and can be was 5 > 2 ≈ 3 > 1 > 6 ≈ 2* > 7 > 8 > 9 > 12 > 10 (Lagrain
solubilized only after reduction of SS bonds. HMW‐GS et al., 2012), which is different to the Tris‐glycine system
belong to the HMW group and are further subdivided originally reported by Laemmli (1970). In 6% Tris‐glycine
 19433638, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cche.10572, Wiley Online Library on [20/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
26 | WIESER ET AL.

gels with an alkaline glycine buffer the HMW‐GS appeared proteins (Lexhaller et al., 2019). Depending on the type,
in the following order: 1 > 2 ≈ 2* ≈ 5 > 3 > 6 > 7 > 14 ≈ other protein groups, such as 2% to 26% of amylase/trypsin‐
17 > 15 ≈ 18 > 8 > 10 ≈ 9 > 12 (Geisslitz et al., 2020). inhibitors, <2% to 6% of avenin‐like proteins, and <2%
Gluten protein types can be isolated from wheat flour to 5% globulins were also present in the respective isolates.
using modified Osborne fractionation to extract the gliadin A closer look at the gluten proteins revealed that co‐
and glutenin fractions, followed by reversed‐phase high‐ mingling of different gluten protein types occurred, for
performance liquid chromatography to separate and enrich example, with substantial relative amounts of LMW‐GS in
the different types according to hydrophobicity (Figure 3) both the α‐ and γ‐gliadin isolates. These results highlighted
(Schalk et al., 2017). Discovery proteomics experiments of the intricate complexity of gluten proteins and their ability
the separated HMW‐GS, LMW‐GS, α‐, γ‐, ω1,2‐, and to bind other proteins and other flour constituents.
ω5‐gliadin isolates from common wheat flours showed that
this two‐step strategy was suitable to obtain the specific
types, with gluten proteins making up 58% to 87% of total 3 | P R I M A R Y ST R U C T U R E O F
GLUTEN PROTEINS

3.1 | Amino acid composition

The amino acid composition of gluten proteins is generally

characterized by high proportions of glutamine (32% to 53%)
and proline (11% to 29%) (Supporting Information:
Figure S1). Both amino acids are important for the biological
function of gluten as storage proteins. In contrast to most
other amino acids, glutamine contains two nitrogen atoms
that supply sufficient nitrogen to the germinating grain.
Proline with its secondary amino group causes kinks within
the secondary protein structure and, therefore, allows a
dense packing of the protein strands in the starchy
endosperm (Tosi, 2012). Moreover, peptide bonds that
contain a proline residue are resistant to most peptidases
and prevent the storage proteins from extensive degradation
by external enzymatic attacks (Simpson, 2001).
Next to glutamine and proline, hydrophobic amino acid
residues such as valine, leucine, isoleucine, and phenyl-
alanine show relatively high proportions (as a sum up to
28%). These amino acids are responsible for the hydropho-
bicity of gluten proteins that prevent the leakage of proteins
out of the grain during germination. Although cysteine
belongs to the minor amino acids (≈2%), it is very important
for the formation of intra‐ and interchain SS bonds and the
resulting functionality of gluten (Köhler et al., 1997).
Amino acid residues with positive (lysine and arginine) or
negative (aspartic acid and glutamic acid) charges are rare
(1.0% to 3.8% and 1.4% to 3.3%, respectively). Further
common features of gluten proteins are their low content of
the essential amino acids lysine (0.3% to 1.1%), methionine
(0.0% to 1.8%), and tryptophan (0.0% to 1.0%). Altogether,
the amino acid composition of gluten proteins is well‐suited
to fulfill their function as storage proteins: a source of
F I G U R E 3 RP‐HPLC chromatograms of the gliadin (a) and nitrogen and amino acids for the germinating grain, tightly
glutenin (b) fractions extracted from the German common wheat packed in the starchy endosperm, resistant to degradation
variety Akteur. α, γ, ω, gliadin types; GS, glutenin subunits; HMW, by external enzymes, and insoluble in water.
high molecular weight; LMW, low molecular weight; RP‐HPLC, HMW‐GS are characterized by a high content of
reversed‐phase high‐performance liquid chromatography. glutamine (32% to 36%), proline (11% to 13%), and
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WIESER ET AL. | 27

additionally glycine (18% to 20%), which together account modern‐day wheat (Pont et al., 2019). Nevertheless, gluten
for approximately 65% of their total composition. More- proteins mostly show 75% to 97% of homology within each
over, HMW‐GS have more tyrosine (5.2% to 5.7%) and less protein type. The proteome of Triticum aestivum currently
phenylalanine (0.3%) compared to the other gluten protein contains 143,219 amino acid sequences in total, but only
types. Differences between x‐ and y‐type HMW‐GS are 379 (less than 0.3%) manually annotated and reviewed
small and mainly present in the content of arginine (1.2% sequences (UniProtKB; August 02, 2021).
vs. 2.7%) and histidine (0.5% vs. 2.4%). ω5‐ and ω1,2‐ A schematic overview of the different sequence
gliadins have extremely unbalanced amino acid composi- domains of gluten protein types was provided by Wieser
tions marked by the highest content of glutamine, proline, et al. (2020). The amino acid sequences of HMW‐GS can
and phenylalanine among gluten proteins, accounting for be divided into three structural domains (Figure 4): a
around 80% of the total composition. ω5‐Gliadins show a nonrepetitive N‐terminal domain A comprising 80 to 105
higher glutamine content (53% vs. 42%), a lower proline residues, a repetitive central domain B of 480 to 700
content (20% vs. 29%), and a similar content of residues, and a nonrepetitive domain C of 40 residues
phenylalanine (9% vs. 8%) compared to ω1,2‐gliadins. (P. R. Shewry et al., 1992). Domains A and C are
Isoleucine (4.3% vs.1.6%), leucine (3.1% vs. 4.0%), and characterized by relatively balanced amino acid composi-
serine (2.9% vs. 5.9%) present further significant differ- tions, including most or all cysteine residues and charged
ences between ω5‐ and ω1,2‐gliadins. All other amino amino acids (glutamic acid and arginine). Domain B
acids are below 2.5% or even missing (cysteine and contains repetitive hexapeptides such as PGQGQQ as a
methionine). The amino compositions of α‐gliadins, backbone with distinct differences between x‐ and y‐types
γ‐gliadins, and LMW‐GS are related and have a relatively (Supporting Information: Figure S2). These repeats are
high content of hydrophobic amino acids (phenylalanine, frequently modified by single amino acid residues and
tyrosine, leucine, and valine) apart from glutamine (32% separated by nonapeptides such as GYYPTSPQQ or
to 36%) and proline (13% to 18%). The higher serine GYYPTSLQQ and the tripeptide GQQ (x‐type), or the
content of LMW‐GS (8.9%), the higher asparagine content hexapeptide HYPASQ (y‐type). In comparison to the x‐
of α‐gliadins (2.6%), and the low tyrosine content of type, y‐type HMW‐GS shows a longer domain A,
γ‐gliadins (0.3%) compared to the other proteins of the including two neighboring cysteine residues, and a shorter
LMW group are characteristic features, respectively. more frequently modified domain B (about 50 repeats vs.
70 repeats). Geisslitz et al. (2020) used a proteomics
workflow with high‐resolution LC‐MS/MS to study
3.2 | Amino acid sequences differences in the amino acid sequences of Bx6 and Bx7
HMW‐GS from common wheat, spelt, and emmer. One
Numerous modifications of the ancestral genes during peptide (WQPGQGQQGY) was specific for Bx7 from
wheat evolution resulted in substitutions, insertions, and common wheat and it allowed a differentiation to Bx6
deletions of single or multiple amino acid residues of from common wheat as well as Bx6 and Bx7 from emmer
gluten proteins that can be used to trace the origins of and spelt.

F I G U R E 4 Schematic representation of different domains (a–c) and segments (I–V) of gluten protein types adapted from Wieser et al.
(2020). The following amino acid sequences were used without signal peptide (UniProtKB identifier in parentheses): HMW‐GS x (Q6R2V1),
HMW‐GS y (Q52JL3), ω5‐gliadin (Q402I5), ω1,2‐gliadin (Q6DLC7), α‐gliadin (Q9M4M5), γ‐gliadin (Q94G91), and LMW‐GS (Q52NZ4). GS,
glutenin subunit; HMW, high molecular weight; LMW, low molecular weight. The numbers at the C‐terminal side of the proteins show the
numbers of amino acids per protein.
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28 | WIESER ET AL.

ω5‐ and ω1,2‐gliadins are composed of sequences that 4 | T H R E E ‐ DIMENSIONAL

almost entirely consist of repetitive units (domain B), STRUCTURE O F G LUTEN
with only short nonrepetitive N‐terminal and C‐terminal PROTEINS
domains A and C (Figure 4). Domains A and C have a
balanced amino acid composition, while the central Three‐dimensional structural elements (secondary and
domain B mainly consists of glutamine, proline, and tertiary structures) of gluten proteins have been pre-
phenylalanine. Typical repetitive units of ω5‐gliadins are dicted from amino acid sequences or determined directly
relatively short (e.g., QQQFP) and repeated up to 50 by different spectroscopic measurements of the whole
times, while ω1,2‐gliadins differ due to a decreased polymer, gluten protein types, single proteins, or
number of up to 30 repetitive units (e.g. QQPQQPFP) synthetic peptides. Examples are shown in Figure 5.
(Supporting Information: Figure S3). The models suggest a stretched structure of HMW‐GS, in
The amino acid sequences of α‐gliadins, γ‐gliadins, particular of the x‐type, while gliadins and LMW‐GS
and LMW‐GS are partly homologous and can be divided have a more compact shape. Regarding HMW‐GS,
into an N‐terminal domain containing segments I and II domains A and C show globular steric structures with
and a C‐terminal domain containing segments III–V α‐helices and β‐sheets (P. R. Shewry et al., 1992). For
(Figure 4) (Kasarda et al., 1984). The N‐terminal domains example, the N‐terminal domain of HMW‐GS 1Dx5 has
(40% to 50% of the total sequence) start with short non‐ been predicted to form a continuous strand of an α‐helix
repetitive sequences (segments Ia), which are character- corresponding to residues 5–32, while that of 1Bx7 forms
istic of each type. Segment Ib exclusively consists of several shorter regions of α‐helices (Köhler et al., 1997).
unique repetitive units such as QPQPFP and PQQPYP An intrachain SS bond stabilizes the structure of domain
repeated up to five times (α‐gliadins), QQPQQPFP A. Domain B is characterized by a stretched conforma-
repeated up to 16 times (γ‐ gliadins), and QQQPPFS tion with repeated β‐turns. These occur in regions, where
repeated up to 13 times (LMW‐GS) (Supporting Informa- the protein backbone abruptly changes direction. Such
tion: Figure S4). Segment II entirely consists of glutamine regions in domain B include four amino acid residues,
(up to 18 residues) and occurs only in α‐gliadins. The consisting of proline, glutamine, and glycine (QPGQ),
C‐terminal domain (segments III–V) presents sequences and form repeated β‐turns, which create a loose spiral
that are nonrepetitive, have less glutamine and proline structure (β‐spiral) that is thought to confer elastic
than the N‐terminal domain, and possess a more properties to the proteins (Kasarda et al., 1994; Tatham
balanced amino acid composition. Segment III presents et al., 1985).
the highest degree of homology in both length and ω‐Gliadins almost entirely contain proline‐rich repet-
composition, while segment IV is partly homologous and itive units, which cause a stretched conformation of the
partly unique for each type. Segment V includes molecule characterized by β‐turns and random coils in
homologous sequences (Va) and short unique sequences between (Purcell et al., 1988; Tatham & Shewry, 1985).
(Vb). There are no detectable α‐helices or β‐sheets or SS bonds,

F I G U R E 5 Models representing the three‐dimensional structure of high‐molecular‐weight glutenin subunits (HMW‐GS) x‐type (a) and
y‐type (b), α‐gliadins (c), γ‐gliadins (d), and low‐molecular‐weight glutenin subunits (LMW‐GS). Proteins were modeled with SWISS‐
MODEL (Waterhouse et al., 2018). The minor constituents ω5‐ and ω1,2‐gliadins are missing, because no sufficient quality homology model
template was available.
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WIESER ET AL. | 29

whereas hydrophobic interactions are important. Accord- alcohol‐soluble oligomers. Free cysteine and glutathione
ing to partially homologous amino acid sequences, naturally occurring in flour have been identified as
α‐gliadins and γ‐gliadins have related secondary and further terminators (Schmid et al., 2017).
tertiary structures. Segment I is characterized by a β‐turn The alcohol‐insoluble glutenin fraction (≈35% of total
conformation similar to that of ω‐gliadins (Tatham & gluten proteins) contains polymers, mainly consisting of
Shewry, 1985). Segment II of α‐gliadins, that is, the poly‐ SS‐linked LMW‐GS and HMW‐GS with MWs ranging
glutamine sequence, is characterized by an α‐helical from around 700,000 to more than 10 million. The largest
conformation. The C‐terminal domains (segments III–V) polymers termed “glutenin macropolymer” (GMP) or
contain considerable proportions of α‐helices and β‐sheet “unextractable polymeric protein” have MWs well in the
structures with small regions of β‐turn and random coil. multimillion range and may belong to the largest protein
The tertiary structure is stabilized by three (α‐ gliadins) aggregates in nature (Don et al., 2003; Wrigley, 1996).
and four (γ‐gliadins) intrachain SS bonds, respectively. GMP may be defined as the large‐size portion of the
Information on LMW‐GS steric structures is rare. Due to glutenin fraction that is insoluble in an aqueous SDS
corresponding sequence segments, they are related to solution. GMP can be prepared from flour after extrac-
those of α‐ and γ‐gliadins. The repetitive segment I is rich tion of other components with a diluted SDS solution
in β‐turns, while segments III–V have globular structures (e.g., 1.5%) followed by centrifugation and decanting of
with elements of α‐helix, β‐turn, β‐sheet, and random the supernatant (Moonen et al., 1982). The GMP‐
coil (Tatham et al., 1987). Three intrachain SS bonds containing gel layer, formed on the surface of the starch
stabilize the steric structure of the C‐terminal domain. pellet, is scraped off, washed with ethanol and water, and
lyophilized. The amount of GMP in common wheat flour
is in the range of 20–40 mg/g of flour (≈3% of gluten
5 | M O L E C U L A R WE I G H T proteins) and is correlated with dough strength and
DISTRIBUTION bread volume (Thanhaeuser et al., 2014; Weegels
et al., 1996). Recent quantitative data for two common
Gluten proteins are characterized not only by their high wheat varieties (Akteur/Winnetou) revealed that GMP
number of individual components and their exceptional consists of 54%/50% LMW‐GS, 31%/39% HMW‐GS, and
amino acid sequences but also by their broad MW around 10% gliadins. The ratios of LMW‐GS to HMW‐GS
distribution in the native state (Koehler & Wieser, 2013; (1.7/1.3) were lower than the ratios found in the total
Southan & MacRitchie, 1999). Monomeric gliadins glutenin fraction (1.9/1.6), indicating enrichment of
(α‐, γ‐, and ω‐gliadins), corresponding to ≈50% of total HMW‐GS in GMP compared to total glutenins (Mueller
gluten proteins, have MWs of approximately 28,000 et al., 2016).
to 55,000. They are alcohol‐soluble and are either devoid The MW distribution of native glutenins has been
of SS bonds (ω‐gliadins) or have intrachain SS bonds recognized as one of the main determinants of dough
(α‐gliadins and γ‐gliadins). In contrast, there are properties and baking performance (P. R. Shewry
apparently no glutenin monomers. et al., 2003). A shift in the distribution curve towards
Apart from monomers, the gliadin fraction contains higher MWs, corresponding to higher GMP content,
alcohol‐soluble oligomers with MWs ranging from 70,000 results in a stronger dough with greater resistance to
to about 700,000. Thus, around 2 to 20 protein units are mixing and increased stability. The greater the average
linked by interchain SS bonds. This subfraction has been length of the polymers, the more they can overlap,
called HMW‐gliadin, aggregated gliadin, or ethanol‐ interacting to form a continuous matrix surrounding the
soluble glutenin and accounts for ≈15% of gluten proteins starch granules in the dough. Three main factors appear
(Huebner & Bietz, 1993; Schmid et al., 2016; P. R. Shewry to govern the MW distribution of glutenins, (a) the
et al., 1983; Southan & MacRitchie, 1999). HMW‐gliadin HMW‐GS/LMW‐GS ratio, (b) the allelic variation at Glu‐
is chemically less well characterized in comparison with 1 loci (e.g., the presence of HMW‐GS 1Dx5 + 1Dy10 vs.
total gliadin and glutenin fractions. After reduction of SS 1Dx2 + 1Dy12) and (c) the proportions of chain termina-
bonds, the following proportions of gluten protein types tors (Lafiandra et al., 1999; MacRitchie, 1999).
were quantitated in HMW‐gliadin isolated from the
German common wheat variety Akteur: 48% LMW‐GS,
18% γ‐gliadins, 13% α‐gliadins, 9% ω1,2‐gliadins, 8% 6 | D I S U L F I D E ST R U C T U R E
HMW‐GS, and 4% ω5‐gliadins (Schmid et al., 2016) It has
been shown that modified gliadins with an odd number SS bonds play a key role in determining the structure of
of cysteine residues act as terminators and stop the gluten proteins. They are formed between thiol (SH‐)
polymerization of HMW‐GS and LMW‐GS resulting in groups of cysteine residues either within one protein
 19433638, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cche.10572, Wiley Online Library on [20/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
30 | WIESER ET AL.

(intrachain) or between more than one protein (interchain). α‐ and γ‐gliadins. Two cysteine residues, located in
Intrachain bonds stabilize the tertiary structure of the segments I and IV, are unique to LMW‐GS and are not
protein, whereas interchain bonds induce the formation of known to build an intrachain SS bond, probably because
protein aggregates. Thus, cysteine is decisive for the of steric hindrance. Instead, they are involved in
structure of gluten proteins, although it belongs to the interchain SS bonds with cysteine residues of modified
minor amino acids (≈2 mol%). SS‐formation via SH‐ gliadins, LMW‐GS, and HMW‐GS.
oxidation of cysteine residues mediated by protein disulfide Both x‐ and y‐type HMW‐GS clearly differ from
isomerase starts shortly after protein synthesis in the lumen LMW‐GS in number and position of cysteine residues
of the endoplasmic reticulum of endosperm cells as an and thus in SS bond formation (Figure 6). x‐Type
integral part of protein folding (Osipova et al., 2012). It is subunits, except subunit 1Dx5, have three cysteine
likely that intrachain bonds form more rapidly than residues in domain A and one in domain C. Two
interchain bonds. The oxidation process continues until residues of domain A form an intrachain SS bond and the
most of the cysteine residues are linked by SS bonds. The other residues form interchain SS bonds with other
importance of SS bonds for dough quality can be HMW‐GS molecules (so‐called head‐to‐tail linkages).
demonstrated by adding reducing agents that result in Subunit 1Dx5 has an additional cysteine residue in
dough weakening and thiol blocking or oxidizing agents domain A and might form another interchain SS bond.
that lead to dough strengthening (Lagrain et al., 2007). y‐Type subunits have five cysteine residues in domain A
ω‐Gliadins are predominantly devoid of cysteine and and one each in domains B and C. Interchain links were
occur as monomers, apart from ωb‐gliadins. Most α‐ and identified for adjacent cysteine residues of domain A,
γ‐gliadins contain six and eight cysteine residues, respec- which are connected in parallel with corresponding
tively, and form three or four homologous intrachain SS residues of another y‐type HMW‐GS. An additional
bonds either within sequence segment III or between cysteine residue is present in domain B, which is linked
segments III and V (Figure 6). They are responsible for the to the cysteine residue located in segment IV of LMW‐
compact three‐dimensional structure of the α‐ and γ‐gliadin GS. In summary, HMW‐ and LMW‐GS fulfill the
molecules. Due to point mutations, some ω‐, α‐, and requirement that allows polymerization with at least
γ‐gliadins (“modified gliadins”) contain an odd number of two cysteine residues per molecule available for inter-
cysteine residues and this leaves one SH group available for chain SS bond formation.
crosslinking to other modified gliadins or to glutenins by The most recent glutenin model suggests a backbone
interchain SS bonds (Lutz et al., 2012). consisting of HMW‐GS linked by head‐to‐tail SS bonds
LMW‐GS contain eight cysteine residues, six of which (Figure 7) (Köhler et al., 1993, Wieser et al., 2014). LMW‐
form three intrachain SS bonds homologous to those of GS align as linear polymers via cysteine residues of

F I G U R E 6 Representation of the disulfide structure of wheat gluten proteins. Nomenclature according to Köhler et al. (1993). GS,
glutenin subunit; HMW, high molecular weight; LMW, low molecular weight.
 19433638, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cche.10572, Wiley Online Library on [20/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
WIESER ET AL. | 31

involving between 2 and 20 units present in HMW‐

gliadin and up to more than 300 units present in GMP.

7 | GL UTEN C HEMISTRY A ND
FU NCT I ON A LI TY

The characteristic molecular structures of gluten proteins

are the basis for the unique physical properties of dough
and the bread‐making quality of wheat. When wheat
flour is mixed with water, hydrated gluten proteins
enable the development of a dough characterized by a
F I G U R E 7 Model of the three‐dimensional glutenin network balanced interrelation of cohesivity, viscosity, extensi-
made up of x‐ and y‐type HMW‐GS and LMW‐GS modified from bility, and elasticity. The gluten network mediates the
Wieser et al. (2014). Disulfide‐linked HMW‐GS are displayed gas‐holding properties of dough during fermentation and
horizontally, and LMW‐GS are attached to this backbone via baking. When the fully developed dough is washed with
intermolecular disulfide bonds. HMW‐GS, high‐molecular‐weight water to remove starch granules and soluble constituents,
glutenin subunit; LMW‐GS, low‐molecular‐weight glutenin a rubber‐like, water‐insoluble gluten mass remains. This
subunit. wet gluten mass is a concentrate of all rheological dough
features and can be stored as so‐called vital gluten after
drying. Vital gluten has many applications not only in
segments I and IV and they are linked to domain B of baking to standardize dough and bread quality but also in
y‐type HMW‐GS. The SS structure of native gluten other food and nonfood products (Day et al., 2006).
proteins, described here, undergoes significant changes The insolubility of gluten proteins in water or salt
during the dough‐making and baking process (Lagrain solution is an essential prerequisite for the formation of a
et al., 2007). For example, most monomers containing gluten network during dough mixing. Gluten isolated
intrachain SS (α‐ and γ‐gliadins) are bound to the from wheat dough binds about twice its own weight of
glutenin polymers via interchain SS bonds upon heating. water due to the high water‐binding capacity of gluta-
Disulfide interchange reactions have been shown to be mine residues (Schopf et al., 2021), but the proteins still
involved in the heat‐induced effects, next to non‐SS remain insoluble. The nonrepetitive sequences that
crosslinks, for example, isopeptide crosslinks, lanthio- contain relatively high proportions of hydrophobic
nine, and lysinoalanine crosslinks (Rombouts et al., 2011). amino acid residues (phenylalanine, leucine, isoleucine,
Similarly, treatment of gluten proteins with high hydro- and valine) and less glutamine apparently prevent
static pressure provokes a shift of α‐ and γ‐gliadins into the protein solubilization.
glutenin fraction, indicating the cleavage of intrachain SS The presence of both hydrophilic and hydrophobic
bonds and their rearrangement into interchain SS bonds sequence segments confers emulsifying properties to
(Kieffer et al., 2007). gluten proteins, especially, to partially hydrolyzed gluten.
Polymerization of HMW‐ and LMW‐GS is stopped by Acid treatment of gluten, for example, increases the
so‐called terminators (Kasarda, 1989). These include free hydrophilic character of gluten proteins via partial
thiols such as glutathione or cysteine as well as proteins deamidation of glutamine to glutamic acid residues and
with an odd number of cysteine residues. To identify enhances the foaming and emulsifying properties.
possible terminators, HMW‐gliadin was partially hydro- Alternatively, enzymatic hydrolysis, using, for example,
lyzed with thermolysin and SS bonds were identified in alcalase or trypsin, produced peptides with emulsifying
the resulting peptides. Apart from 26 peptides, that properties (Joye & McClements, 2014). These so‐called
contained SS bonds with known gliadin and glutenin hydrolyzed wheat proteins are frequently used as
crosslinks, 15 peptides with SS bonds unique to HMW‐ additives for food (e.g., soup and ice cream) and
gliadin were detected. They include bonds between cosmetics (e.g., soap and shampoo) and show considera-
modified gliadins and glutenins such as α‐gliadin/ ble differences regarding MW distribution, solubility, and
HMW‐GS, γ‐gliadin/HMW‐GS, and ω‐gliadin/LMW‐GS hydrophilicity/hydrophobicity (Gabler & Scherf, 2020).
as well as bonds between glutathione and HMW‐GS Apart from insolubility, the cohesivity of gluten
(Schmid et al., 2017). In conclusion, glutenin polymeri- proteins is important for gluten network formation and
zation occurring after synthesis is partially interrupted by stability. Cohesivity results from strong binding forces
terminators. The result is a complex mixture of SS links, between proteins. The gluten structure determined by
 19433638, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cche.10572, Wiley Online Library on [20/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
32 | WIESER ET AL.

covalent SS bonds is superimposed by noncovalent glutenins are both cohesive and elastic and are thus
hydrogen bonds, ionic bonds, and hydrophobic bonds, responsible for dough strength and elasticity. For ease of
which are important for gluten aggregation. Glutamine understanding, the gluten network represents a two‐
residues are not only responsible for water binding, but component glue, in which gliadins can be seen as a
also for interchain hydrogen bonds. The dough weaken- “plasticizer” or “solvent” for glutenins. An appropriate
ing effect of hydrogen‐bond breaking agents such as urea ratio of both fractions is, therefore, essential to impart the
provides experimental evidence for the presence of viscoelastic dough properties required to achieve a high‐
hydrogen bonds between gluten proteins. Vice versa, quality end product. A high ratio of gliadins to glutenins
heavy water (D2O) has a dough strengthening effect (e.g., 2.4 to 3.1) leads to less viscous and more extensible
compared to water (H2O) because deuterium bonds are (soft) doughs, whereas a low ratio (e.g., 1.7 to 2.2)
considerably stronger than hydrogen bonds (Inda & generates highly viscous and less extensible (strong)
Rha, 2007). Although charged amino acid residues are doughs (Kieffer et al., 1998; Wieser & Kieffer, 2001).
rare, ionic bonds are also important for interactions
between gluten proteins. Increasing the number of ionic
bonds by adding salts (e.g., NaCl) is known to strengthen 8 | F U T U R E P ER S P E C T I V E S
dough due to the promotion of ordered interactions of
glutenins with gliadins (Ukai et al., 2008). Dipolar ions As outlined above, significant discoveries have been
such as amino acids or dicarboxylic acids also strengthen made by a number of dedicated research groups world-
gluten isolated from the dough by acting as spacers and wide to elucidate the qualitative composition of wheat
the formation of additional ionic bonds within the gluten gluten proteins since the pioneering work of T. B.
network. Hydrophobic bonds involving the side chains of Osborne in the 1900s (Osborne, 1907). Despite this
phenylalanine, leucine, isoleucine, and valine, can also progress, there are still many unresolved questions
significantly contribute to the stabilization of gluten related to the complex chemistry of wheat gluten
protein structures and are particularly important when proteins and their dynamic interactions. While SS bonds
the dough is heated during baking. are an important determinant of gluten structure and
Elasticity, as a particularly important physical prop- functionality, not all positions and redox states of SS
erty of wheat dough, has been ascribed to the glutenin bonds have been identified on a molecular level. Even
fraction, and in particular, to domain B of HMW‐GS. The less knowledge is available on how different processing
regularly repeated sequence unit QPGQ generates techniques from milling to dough‐making and baking or
β‐turns that are linked by GQ and form a loose β‐spiral extrusion influence SH/SS exchange reactions and other
similar to the repetitive sequences of elastin, an elastic stabilizing forces, particularly hydrogen bonds. Most
protein of the connective tissue. Dough mixing has been research activities are dedicated to common wheat, some
shown to increase α‐helix, β‐turn, and β‐sheet secondary on durum wheat, and only a few to other wheat species
structures, indicating that the proteins form a more such as spelt, emmer, and einkorn. Renewed interest in
ordered conformation (Seabourn et al., 2008). utilizing these species for specialty products, will likely
A different study postulated that interchain hydrogen spark further investigations into the qualitative composi-
bonds between domains B of HMW‐GS contribute to tion of gluten proteins from these species. With modern
gluten elasticity. Results of infrared spectroscopy suggest omics workflows being increasingly adopted in cereal
that there is an equilibrium between regions forming chemistry, new approaches are now available to gain
interchain hydrogen bonds (“trains,” β‐sheets) and those insights into wheat protein structure at unprecedented
without interchain bonds (“loops,” β‐turns). Stretching sensitivity, accuracy, and speed. Deep learning algo-
results in the conversion of β‐turn to β‐sheet in relation rithms, like AlphaFold (Jumper et al., 2021), provide
to the force applied. In the case of full elastic recoil, the exciting new opportunities in predicting highly accurate
original loop‐train equilibrium should be reinstated, but three‐dimensional protein structures, even in the absence
experimental evidence suggests incomplete elastic recoil of similar structural models, as is currently the case for
and buildup of β‐sheets with each extension/relaxation wheat proteins. Taken together, these novel tools will
cycle (Wellner et al., 2005). enhance our understanding of gluten protein structure
Gluten proteins are fundamental contributors to the and how it is affected by different genetic and environ-
viscosity, extensibility, and elasticity of dough, but the mental factors as well as processing. This knowledge will
specific functions of gliadins and glutenins are divergent. be essential to adapt wheat production to elevated CO2
Hydrated gliadins have little elasticity and are less levels in the atmosphere and to climate change with its
cohesive than glutenins and mainly determine dough associated increasingly extreme weather conditions.
viscosity and extensibility. In contrast, hydrated Selecting and breeding more resilient wheat varieties
 19433638, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cche.10572, Wiley Online Library on [20/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
WIESER ET AL. | 33

with favorable processing properties will be a key Geisslitz, S., America, A. H. P., & Scherf, K. A. (2020). Mass
element to help ensure nutrition and food security spectrometry of in‐gel digests reveals differences in amino acid
worldwide. sequences of high‐molecular‐weight glutenin subunits in spelt
and emmer compared to common wheat. Analytical and
Bioanalytical Chemistry, 412, 1277–1289.
AUTHOR CONTRIBUTIONS
He, J., Penson, S., Powers, S. J., Hawes, C., Shewry, P. R., & Tosi, P.
Writing—original draft: Herbert Wieser and Katharina (2013). Spatial patterns of gluten protein and polymer
Anne Scherf. Writing—review and editing: Peter Koehler. distribution in wheat grain. Journal of Agricultural and Food
Chemistry, 61, 6207–6215.
ACKNOWLEDGMENTS Huebner, F. R., & Bietz, J. A. (1993). Improved chromatographic
This study is supported by funds from the Federal separation and characterization of ethanol‐soluble wheat
Ministry of Food and Agriculture (BMEL) based on a proteins. Cereal Chemistry, 70, 506–511.
decision of the Parliament of the Federal Republic of Inda, A., & Rha, C. (2007). Dynamic viscoelastic behavior of wheat
Germany via the Federal Office for Agriculture and Food gluten: The effects of hydrogen bonding modification by urea
and deuterium oxide. Journal of Texture Studies, 22, 393–411.
(BLE) under the innovation support program, project
Joye, I. J., & McClements, D. J. (2014). Emulsifying and emulsion‐
BigBaking (2818404B18). Open access funding enabled stabilizing properties of gluten hydrolysates. Journal of
and organized by Projekt DEAL. Agricultural and Food Chemistry, 62, 2623–2630.
Juhász, A., Belova, T., Florides, C. G., Maulis, C., Fischer, I., Gell, G.,
CONFLI CT OF I NTER EST Birinyi, Z., Ong, J., Keeble‐Gagnere, G., Maharajan, A., Ma, W.,
The authors declare no conflict of interest. Gibson, P., Jia, J., Lang, D., Mayer, K. F. X., Spannagl, M.,
International Wheat Genome Sequencing Consortium, Tye‐
ORCID Din, J. A., Appels, R., & Olsen, O.‐A. (2018). Genome mapping
of seed‐borne allergens and immunoresponsive proteins in
Peter Koehler http://orcid.org/0000-0001-7766-9181
wheat. Science Advances, 4, eaar8602.
Katharina A. Scherf http://orcid.org/0000-0001-
Juhász, A., & Gianibelli, M. C. (2006). Low‐molecular‐weight
8315-5400 glutenin subunits: Insights into this abundant subunit group
present in glutenin polymers. In C. Wrigley, F. Bekes, & W.
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Changes in protein secondary structure during gluten