Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state

Abstract

The structure of the central repetitive domain of high molecular weight (HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type I1 p-turns at positions predicted from the model studies; type I1 p-turns occurred at QPGQ and QQGY sequences and type I p-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of p-turns. The type I p-turns are lost when the protein is dried on a solid surface, probably by conversion to type I1 p-turns. The homogeneous type I1 p-turn distribution is compatible with the formation of a P-spiral structure, which provides the protein with elastic properties. The p-turns and thus the P-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.Keywords: elastic properties; repetitive proteins; structural characterization; wheat gluten proteins The central repetitive domain of high molecular weight proteins forms 60-80% of their amino acid sequence and is built from the consensus peptides PGQGQQ, GYYPTS(P/L)QQ, and GQQ Shewry et al., 1994). Its structural organization may consist of a series of p-turns that organize in a @spiral structure (Tatham et al., 1984;Shewry et al., 1994); this idea is supported by scanning tunneling microscopy images of HMW Dx5 (Miles et al., 1991). The proposed &spiral structure for HMW proteins is similar to that of elastin, where it forms the basis for the molecule's elastic properties (Uny, 1993). The structural analogy between HMW proteins and elastin suggests that the HMW proteins might have elastic properties as well; however, no Reprint requests to: G.T. Robillard, Department of Biochemistry and Biophysical Chemistry and the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; e-mail: G.T.Robillard@chem.rg.nl.'Present address: Gist Brocades N.V. Postbus 1, 2600 MA Delft, The Netherlands.Abbreviurions: FITR, fourier transform infrared; HMW, high molecular weight; IPTG, isopropyl-P-D-thiogalactopyranoside. detailed structural characterization of the HMW repetitive domain has been performed. In the companion paper (van Dijk et al., 1997), we described the structure of the consensus sequences of the repetitive domain using cyclic and linear peptides. We showed that they are highly structured and contain a mixture of type I and I1 p-turns that are stabilized by mu...

Size and shape of the repetitive domain of high molecular weight wheat gluten proteins. I. Small‐angle neutron scattering

“…The SDS band was found at Ϸ26.5 kDa. 16 The yield of dB4 after Ni 2ϩ -agarose purification was roughly 5 mg/L cell culture, as determined by absorption. The purities of dB4 after each purification step were comparable with those obtained for dB1.…”

“…A representative part of the central repetitive domain of HMW Dx5 was amplified by polymerase chain reaction (PCR) and subcloned in pSk Ϫ as previously described by van Dijk et al 16 The location and length of dB1 in the DNA sequence is shown schematically in Figure 1A. To obtain concatemers of dB1, Bg/II and BamHI restriction sites were introduced, by PCR, at the 5Ј and 3Ј end of dB1, respectively.…”

“…experimentally. 15,16,21 Their existence was suggested for peptide analogues of the central domain that were found to stabilize a ␤-spiral consisting of at least 30 residues. 15 In addition, it is well-established that the repetitive domains consist of ␤-turns.…”

“…15 In addition, it is well-established that the repetitive domains consist of ␤-turns. 12,13,15,16,19,21,41 The number of ␤-turns present was estimated to be 27 in dB1, and thus 108 in dB4. 16 The flexibility of the cylinder, in combination with the abundant ␤-turns, makes a highly ordered, spiral structure with uniform radius and pitch very unlikely.…”

“…1,2,11 The large central repetitive domain consists of ␤-turns, and was proposed to form a "supersecondary structure" of an elongated, so-called, ␤-spiral. 5,7,8,9,[12][13][14][15][16] The ␤-spirals may contribute to gluten elasticity. The precise molecular basis for the elastic properties of gluten and dough is not known, but is thought to involve contributions from the structures of the individual subunits and their interactions via noncovalent and covalent bonds.…”