Equilibrium unfolding CD studies of bovine ?-lactoglobulin and its 14-52 fragment at acidic pH
1999; Wiley; Volume: 49; Issue: 6 Linguagem: Inglês
10.1002/(sici)1097-0282(199905)49
ISSN1097-0282
AutoresLaura Ragona, L. Confalonieri, Lucia Zetta, C. G. de Kruif, Stefano Mammi, Evaristo Peggion, Renato Longhi, Henriette Molinari,
Tópico(s)Protein Structure and Dynamics
ResumoBovine β-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this β-barrel protein has a surprisingly high α-helical preference, which was retained for short fragments. Cooperative transitions from the native β-sheet to α-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high α-helical preference, it has been proposed that non-native α-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37°C, revealed an apparent two-state transition, and afforded no evidence of non native α-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the β to α structure, provoked by organic solvents in the intact protein. © 1999 John Wiley & Sons, Inc. Biopoly 49: 441–450, 1999
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