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This work follows a previous article that addressed the role of disulfide bonds in the behavior of the 1Dx5 subunit upon hydration. Here the roles of nonrepetitive terminal domains present and the length of the central repetitive domain in the hydration of 1Dx5 are investigated. This was achieved by comparing the hydration behavior of suitable model samples determined by ¹³C- and ¹H-NMR: an alkylated 1Dx5 subunit (alk1Dx5), a recombinant 58-kDa peptide corresponding to the central repetitive domain of 1Dx5 (i.e., lacking the terminal domains), and two synthetic peptides (with 6 and 21 amino acid residues) based on the consensus repeat motifs of the central domain. The ¹³C cross-polarization and magic angle spinning (MAS) experiments recorded as a function of hydration gave information about the protein or peptide fractions resisting plasticization. Conversely, ¹³C single pulse excitation and ¹H-MAS gave information on the more plasticized segments. The results are consistent with the previous proposal of a hydrated network held by hydrogen-bonded glutamines and possibly hydrophobic interactions. The nonrepetitive terminal domains were found to induce water insolubility and a generally higher network hindrance. Shorter chain lengths were shown to increase plasticization and water solubility. However, at low water contents, the 21-mer peptide was characterized by higher hindrance in the megahertz and kilohertz frequency ranges compared to the longer peptide; and a tendency for a few hydrogen-bonded glutamines and hydrophobic residues to remain relatively hindered was still observed, as for the protein and large peptide. It is suggested that this ability is strongly dependent on the peptide primary structure. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 65: 158–168, 2002

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The ¹³C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (~40 and ~65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, ¹³C single pulse excitation and ¹H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by ¹³C T1 and ¹H T1ρ, T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 487–498, 2002

This work describes the application of ¹H magic angle spinning (MAS) nmr to the study of hydrated 1Dx5 wheat high-Mr 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 ¹H 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 ¹³C/¹H 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, Q1 and Q2, and these were characterized in terms of possible conformation and relative dynamics, with the basis of comparison between the single 90° spectrum and the Carr-Purcel-Heiboom-Gill (CPMG) spectrum. The Q1 residues are proposed to be situated in protein segments that adopt the β-sheet conformation and that remain relatively hindered, possibly by hydrogen bonds involving the glutamine amide groups. On the other hand, Q2 residues are proposed to be situated in a more mobile environment, adopting a looser conformation, possibly a β-turn conformation. Based on the proximity of the Q2 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. © 2000 John Wiley & Sons, Inc. Biopoly 58: 33–45, 2001

We have recorded the ¹³C CP-MAS and DD-MAS nmr spectra of dry and hydrated barley storage protein, C-hordein, as a model for wheat S-poor prolamins, together with those of model synthetic peptides (Pro)2(Gln)6(I) and (Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln)3(II) under dry or hydrated conditions. The spectral features of C-hordein as well as these peptides were appreciably different from each other depending on the extent of hydration, reflecting different domains that adopt different types of conformations as well as dynamics. In particular, considerable proportions of the peak intensities were lost in the CP-MAS spectra, and well-resolved ¹³C-nmr signals emerged in DD-MAS nmr spectra owing to acquisition of molecular motions by swelling. It was shown that local β-turn or (Pro)n type II conformation is more preferable for individual Pro residues and β-sheet type conformation is dominant for individual Gln residues in the dry and hydrated systems. In addition, two types of Gln environments are originated in C-hordein that differ in their mobility. Further, ¹³C spin-lattice relaxation times (T1's) of C- hordein and peptide II were reduced by more than one order of magnitude by hydration, reflecting the presence of well-swollen molecular chains. In contrast, theT1 values of peptide I upon hydration remained one third of those in the dry state. Carbon-resolved proton spin-lattice relaxation times in the rotating frame (T1ρ's) were also decreased by about 50% upon hydration, although these parameters were less sensitive as compared to T1 values. In addition, the ¹³C-nmr signals of the aromatic side chain of Phe residues disappeared on hydration owing to interference between the frequency of the acquired flip-flop motion and the proton decoupling frequency. This information gives a new insight into establishing the structural properties of the studied protein system. A model may be put forward for a gel-type structure in which the more rigid part of the system involves intermolecular hydrogen-bonded Gln side chains as well as some hydrophobic “pockets” involving Pro and Phe residues. The liquid-like domain is characterized by considerable backbone and side-chain motion as well as rapid ring-puckering motion in Pro residues. © 1997 John Wiley & Sons, Inc.