Deng Y, Smith D L
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, USA.
Biochemistry. 1998 May 5;37(18):6256-62. doi: 10.1021/bi972711o.
Three unfolding domains in rabbit muscle aldolase destabilized in 3 M urea have been identified from their unfolding rate constants (0.10, 0.036, and 0.0064 min-1). The populations of folded and various, partially unfolded forms were determined by amide hydrogen exchange and mass spectrometry. Results of this study show that unfolding domains may include multiple, noncontiguous segments of the backbone and that different regions of helices may belong to different unfolding domains. In addition, these results show that the domain unfolding most rapidly is located distant from the subunit binding surfaces and has the greatest access to the denaturant. The bimodal intermolecular distributions of deuterium found in this study show that unfolding of these domains is cooperative. It is proposed that these unfolding domains are correlated with local energy minima in the free-energy folding surface of aldolase. In addition to the three unfolding domains, there are three short segments that do not unfold in 3 M urea. These segments, which are located in the subunit binding surface, identify the most stable regions of aldolase. This study also demonstrates that it is now possible to identify and characterize unfolding domains in relatively large (Mr 158 000) proteins.
已根据解折叠速率常数（0.10、0.036和0.0064 min⁻¹）鉴定出兔肌肉醛缩酶中在3 M尿素中不稳定的三个解折叠结构域。通过酰胺氢交换和质谱法测定了折叠形式和各种部分解折叠形式的比例。这项研究的结果表明，解折叠结构域可能包括主链的多个不连续片段，并且螺旋的不同区域可能属于不同的解折叠结构域。此外，这些结果表明，解折叠最快的结构域远离亚基结合表面，并且与变性剂的接触最多。本研究中发现的氘的双峰分子间分布表明这些结构域的解折叠是协同的。有人提出，这些解折叠结构域与醛缩酶自由能折叠表面的局部能量最小值相关。除了这三个解折叠结构域，还有三个短片段在3 M尿素中不解折叠。这些片段位于亚基结合表面，确定了醛缩酶最稳定的区域。这项研究还表明，现在有可能在相对较大（Mr 158 000）的蛋白质中鉴定和解折叠结构域进行表征。
