Four-state equilibrium unfolding of an scFv antibody fragment

被引:18
作者
Pedroso, I [1 ]
Irún, MP [1 ]
Machicado, C [1 ]
Sancho, J [1 ]
机构
[1] Univ Zaragoza, Fac Ciencias, Dept Bioquim & Biol Mol & Celular, E-50009 Zaragoza, Spain
关键词
D O I
10.1021/bi025742e
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein, concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I-1) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I-2), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I-1, I-1-I-2, and I-2-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I-1 one, representing the 'relevant' conformational. stability of the scFv, is small.
引用
收藏
页码:9873 / 9884
页数:12
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