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

¹ Consiglio Nazionale delle Richerche (CNR), Istituto di Chimica del Riconoscimento Molecolare, c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
² Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma "Tor Vergata," 00133 Rome, Italy
³ Instituto Nazionale Fisica della Materia (INFM), Sez. B, Rome, Italy

(RECEIVED March 23, 2004; FINAL REVISION May 14, 2004; ACCEPTED May 18, 2004)

Dystroglycan (DG) is an adhesion complex, expressed in a wide variety of tissues, formed by an extracellular and a transmembrane subunit, -DG and -DG, respectively, interacting noncovalently. Recently, we have shown that the recombinant ectodomain of -DG, -DG(654–750), behaves as a natively unfolded protein, as it is able to bind the C-terminal domain of -DG, while not displaying a defined structural organization. We monitored the effect of a commonly used denaturing agent, the anionic detergent sodium dodecylsulphate (SDS), on -DG(654–750) using a number of biophysical techniques. Very low concentrations of SDS (2 mM) affect both tryptophan fluorescence and circular dichroism of -DG, and significantly perturb the interaction with the -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 -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 -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 -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 -DG ectodomain is an interesting model system.

Keywords: dystroglycan; SDS; NMR spectroscopy; circular dichroism; fluorescence; natively unfolded protein

Abbreviations: DG, dystroglycan • -DG, -dystroglycan • -DG, -dystroglycan • Trx, thioredoxin • NMR, nuclear magnetic resonance • HSQC, heteronuclear single-quantum coherence • CD, circular dichroism • SDS, sodium dodecylsulphate

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04762504.

Reprint requests to: Andrea Brancaccio, Istituto di Chimica del Ricono-scimento Molecolare, c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy; e-mail: andrea.brancaccio{at}icrm.cnr.it; fax: +39-06-3053598.

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