Lakshmikanth G S, Sridevi K, Krishnamoorthy G, Udgaonkar J B
Department of Chemical Sciences, Tata Institute of Fundamental Research Mumbai 400005, India.
Nat Struct Biol. 2001 Sep;8(9):799-804. doi: 10.1038/nsb0901-799.
Coincidental equilibrium unfolding transitions observed by multiple structural probes are taken to justify the modeling of protein unfolding as a two-state, N <==> U, cooperative process. However, for many of the large number of proteins that undergo apparently two-state equilibrium unfolding reactions, folding intermediates are detected in kinetic experiments. The small protein barstar is one such protein. Here the two-state model for equilibrium unfolding has been critically evaluated in barstar by estimating the intramolecular distance distribution by time-resolved fluorescence resonance energy transfer (TR-FRET) methods, in which fluorescence decay kinetics are analyzed by the maximum entropy method (MEM). Using a mutant form of barstar containing only Trp 53 as the fluorescence donor and a thionitrobenzoic acid moiety attached to Cys 82 as the fluorescence acceptor, the distance between the donor and acceptor has been shown to increase incrementally with increasing denaturant concentration. Although other probes, such as circular dichroism and fluorescence intensity, suggest that the labeled protein undergoes two-state equilibrium unfolding, the TR-FRET probe clearly indicates multistate equilibrium unfolding. Native protein expands progressively through a continuum of native-like forms that achieve the dimensions of a molten globule, whose heterogeneity increases with increasing denaturant concentration and which appears to be separated from the unfolded ensemble by a free energy barrier.
通过多种结构探针观察到的巧合平衡展开转变被用来证明将蛋白质展开建模为两态、N⇌U的协同过程是合理的。然而，对于许多经历明显两态平衡展开反应的蛋白质来说，在动力学实验中检测到了折叠中间体。小蛋白质巴氏杆菌抑制因子就是这样一种蛋白质。在这里，通过时间分辨荧光共振能量转移（TR-FRET）方法估计分子内距离分布，对巴氏杆菌抑制因子平衡展开的两态模型进行了严格评估，其中荧光衰减动力学通过最大熵方法（MEM）进行分析。使用仅含有Trp 53作为荧光供体和连接到Cys 82的硫代硝基苯甲酸部分作为荧光受体的巴氏杆菌抑制因子突变体形式，已表明供体和受体之间的距离随着变性剂浓度的增加而逐渐增加。尽管其他探针，如圆二色性和荧光强度，表明标记的蛋白质经历两态平衡展开，但TR-FRET探针清楚地表明是多态平衡展开。天然蛋白质通过一系列类似天然的形式逐渐扩展，达到熔球状态的尺寸，其异质性随着变性剂浓度的增加而增加，并且似乎通过自由能垒与未折叠的整体分离。
