Van Dael H, Haezebrouck P, Pardon E, Joniau M
Interdisciplinary Research Center, Kortrijk, Belgium.
Eur Biophys J. 1997;25(3):171-9. doi: 10.1007/s002490050029.
LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca(2+)-binding site and helix C) of bovine alpha-lactalbumin has been inserted. The equilibrium unfolding of this hybrid protein has been examined by circular dichroism and tryptophan fluorescence techniques. The reversible denaturation process induced by temperature or by addition of chemical denaturant is three-state in the case of apo-LYLA1 and two-state in the presence of Ca2+. The Ca(2+)-bound form of the chimera exhibits higher stability 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 conditions. Combination of circular dichroism with bis-ANS fluorescence experiments has permitted us to characterize the acid state of LYLA1 as a molten globule. Furthermore our results strongly suggest the presence of multiple denatured states depending on external conditions.
LYLA1是一种嵌合蛋白，主要由源自人溶菌酶的残基组成，但其中插入了牛α-乳白蛋白的中心部分（Ca(2+)结合位点和螺旋C）。已通过圆二色性和色氨酸荧光技术研究了这种杂合蛋白的平衡去折叠。在脱辅基LYLA1的情况下，由温度或添加化学变性剂诱导的可逆变性过程是三态的，而在存在Ca2+的情况下是二态的。嵌合体的Ca(2+)结合形式表现出比野生型溶菌酶和α-乳白蛋白更高的稳定性。然而，脱辅基形式的稳定性介于亲本分子之间。脱辅基LYLA1的去折叠涉及一个中间状态，在各种实验条件下该中间状态以不同程度出现。圆二色性与双-ANS荧光实验的结合使我们能够将LYLA1的酸性状态表征为熔球态。此外，我们的结果强烈表明，根据外部条件存在多种变性状态。
