Conformational polymorphism of high Mr glutenin subunits detected by transverse urea gradient gel electrophoresis
1999
Lafiandra, D. | Turchetta, T. | D'Ovidio, R. | Anderson, O.D. | Facchiano, A.M. | Colonna, G.
Unfolding behaviour of different allelic variants of high Mr glutenin subunits has been studied by transverse urea gradient gel electrophoresis, a system in which a gradient of urea (from 0 to 8 M) is generated perpendicularly to the direction of migration. A different unfolding pattern is observed between x- and y-type subunits. The former type of subunits generally present a broad unfolding pattern without any clear transition, as urea concentration increases, suggesting the existence of several conformational intermediates. On the contrary most of the y-type subunits present an unfolding pattern showing an abrupt decrease in mobility, with a single inflection point at the midpoint of the transition indicative of a protein unfolding in a single, cooperative, two-state transition. Native and bacterial expressed subunits, differing for the size of the repetitive domain, were used to assess the role of variation in size of such region on unfolding behaviour. Free energy values associated to unfolding process were calculated for most of the y-type subunits presenting a two state transition. In particular, different values were found for subunit Dy10 and Dy12, with the former being larger than the latter, suggesting a different stability of the two subunits. Possible implications for qualitative differences associated to allelic subunits are also discussed.
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