Equilibrium unfolding CD studies of bovine beta-lactoglobulin and its 14-52 fragment at acidic pH

Bovine beta-lactoglobulin represents an interesting example of context-depe ndent secondary structure induction. In fact, secondary structure predictio ns indicated that this beta-barrel protein has a surprisingly high alpha-he lical preference, which was retained for short fragments. Cooperative trans itions from the native beta-sheet to alpha-helical structures were addition ally induced by organic solvents, in particular trifluoroethanol. As a resu lt of this high alpha-helical preference, it has been proposed that non-nat ive alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better under-standing of the processes that underlie conformational plasticity in this protein, CD measurements i n 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 3 7 degrees C, revealed an apparent two-state transition, and afforded no evi dence of non native alpha-helical inlet-mediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with highe r molar concentration urea. lend to partial misfolding. A 29-mer peptide co vering the region of strands strands a and b of the intact protein, charact erized by the presence of 4/3 heptad repeats, was synthesized and studied b y CD in the presence of different solvents. On the basis of the obtained re sults, a mechanism was proposed to explain the structural transition from t he beta to alpha structure, provoked by organic solvents in the intact prot ein. (C) 1999 John Wiley & Sons, Inc.