CONFORMATIONAL STABILITY OF LYLA1, A SYNTHETIC CHIMERA OF HUMAN LYSOZYME AND BOVINE ALPHA-LACTALBUMIN

LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca2+-binding site a nd helix C) of bovine alpha-lactalbumin has been inserted. The equilib rium unfolding of this hybrid protein has been examined by circular di chroism and tryptophan fluorescence techniques. The reversible denatur ation process induced by temperature or by addition of chemical denatu rant is three-state in the case of apo-LYLA1 and two-state in the pres ence of Ca2+. The Ca2+-bound form of the chimera exhibits higher stabi lity than both wild-type lysozyme and alpha-lactalbumin. The stability of the apo-form, however, is intermediate between that of the parent molecules. Unfolding of apo-LYLA1 involves an intermediate state that becomes populated to a different extent under various experimental con ditions. Combination of circular dichroism with bis-ANS fluorescence e xperiments has permitted us to characterize the acid state of LYLA1 as a molten globule. Furthermore our results strongly suggest the presen ce of multiple denatured states depending on external conditions.