期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：45
页码：15893-15898
DOI：10.1073/pnas.0403979101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The molecular mechanism by which the homodimeric enzyme Cu/Zn superoxide dismutase (SOD) causes neural damage in amytrophic lateral sclerosis is yet poorly understood. A striking, as well as an unusual, feature of SOD is that it maintains intrasubunit disulfide bonds in the reducing environment of the cytosol. Here, we investigate the role of these disulfide bonds in folding and assembly of the SOD apo protein (apoSOD) homodimer through extensive protein engineering. The results show that apoSOD folds in a simple three-state process by means of two kinetic barriers: 2D{rightleftharpoons}2M{rightleftharpoons}M2. The early predominant barrier represents folding of the monomers (M), and the late barrier the assembly of the dimer (M2). Unique for this mechanism is a dependence of protein concentration on the unfolding rate constant under physiological conditions, which disappears above 6 M Urea where the transition state for unfolding shifts to first-order dissociation of the dimer in accordance with Hammond-postulate behavior. Although reduction of the intrasubunit disulfide bond C57-C146 is not critical for folding of the apoSOD monomer, it has a pronounced effect on its stability and abolishes subsequent dimerization. Thus, impaired ability to form, or retain, the C57-C146 bond in vivo is predicted to increase the cellular load of marginally stable apoSOD monomers, which may have implications for the amytrophic lateral sclerosis neuropathology.
关键词：protein folding ; protein stability ; disulfide bond ; transition-state shifts ; protein engineering
