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.

Bozzi, M., Di Stasio, E., Cicero, D.o., Giardina, B., Paci, M., Brancaccio, A. (2004). The effect of an ionic detergent on the natively unfolded beta-dystroglycan ectodomain and on its interaction with alpha-dystroglycan. PROTEIN SCIENCE, 13(9), 2437-2445 [10.1110/ps.04762504].

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

CICERO, DANIEL OSCAR;PACI, MAURIZIO;
2004-01-01

Abstract

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.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10
English
Con Impact Factor ISI
Circular dichroism; Dystroglycan; Fluorescence; Natively unfolded protein; NMR spectroscopy; SDS
Bozzi, M., Di Stasio, E., Cicero, D.o., Giardina, B., Paci, M., Brancaccio, A. (2004). The effect of an ionic detergent on the natively unfolded beta-dystroglycan ectodomain and on its interaction with alpha-dystroglycan. PROTEIN SCIENCE, 13(9), 2437-2445 [10.1110/ps.04762504].
Bozzi, M; Di Stasio, E; Cicero, Do; Giardina, B; Paci, M; Brancaccio, A
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/30367
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