The effect of an ionic detergent on the natively unfolded β-dystroglycan ectodomain and on its interaction with α-dystroglycan

Dystroglycan (DG) is an adhesion complex, expressed in a wide variety of tissues, formed by an extracellular and a transmembrane subunit, alpha-DG and beta-DG, respectively, interacting noncovalently. Recently, we have shown that the recombinant ectodomain of beta-DG, beta-DG(654-750), behaves as a natively unfolded protein, as it is able to bind the C-terminal domain of alpha-DG, while not displaying a defined structural organization. We monitored the effect of a commonly used denaturing agent, the anionic detergent sodium dodecyl-sulphate (SIDS), on beta-DG(654-750) using a number of biophysical techniques. Very low concentrations of SDS (less than or equal to2 mM) affect both tryptophan fluorescence and circular dichroism of beta-DG, and significantly perturb the interaction with the alpha-DG subunit as shown by solid-phase binding assays and fluorescence titrations in solution. This result confirms, as recently proposed for natively unfolded proteins, that beta-DG(654-750) exists in a native state, which is crucial to fulfill its biological function. Two-dimensional NMR analysis shows that SDS does not induce any evident conformational rearrangement within the ectodomain of beta-DG. Its first 70 amino acids, which show a lower degree of mobility, interact with the detergent, but this does not change the amount of secondary structure, whereas the highly flexible and mobile C-terminal region of beta-DG(654-750) remains largely unaffected, even at a very high SDS concentration (up to 50 mM). Our data indicate that SDS can be used as a useful tool for investigating natively unfolded proteins, and confirm that the beta-DG ectodomain is an interesting model system.