Manetto Gaetano D, Grasso Domenico M, Milardi Danilo, Pappalardo Matteo, Guzzi Rita, Sportelli Luigi, Verbeet Martin P, Canters Gerard W, La Rosa Carmelo
Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125 (CT), Italy.
Chembiochem. 2007 Nov 5;8(16):1941-9. doi: 10.1002/cbic.200700214.
The role played by the alpha-helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix-depleted azurin variant produced by site-directed mutagenesis. The protein structure was investigated by CD, 1D (1)H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV-visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the Cu(II) site. The effects of the alpha-helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature. The results show that, in the absence of the alpha-helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60% with respect to the wild-type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small beta-sheet cupredoxins, the results as a whole support the hypothesis that the presence of the alpha-helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved beta-sheet core provides a scaffold for cooperative folding of the wild-type protein.
通过研究定点诱变产生的缺乏α-螺旋的天青蛋白变体，探讨了α-螺旋在决定天青蛋白的结构、稳定性和去折叠机制中所起的作用。通过圆二色光谱（CD）、一维（1）H核磁共振（NMR）、荧光光谱测量和分子动力学（MD）模拟研究蛋白质结构，同时进行电子顺磁共振（EPR）、紫外可见光谱和循环伏安实验以研究Cu（II）位点的几何结构和性质。通过差示扫描量热法（DSC）、升温过程中的紫外/可见光谱和荧光测量，确定α-螺旋缺失对蛋白质热稳定性和去折叠途径的影响。结果表明，在没有α-螺旋片段的情况下，蛋白质的整体结构得以维持，只有铜位点略有改变。相比之下，与野生型天青蛋白相比，该蛋白质的稳定性降低了约60%。此外，突变型天青蛋白的去折叠途径涉及可检测中间体的存在。与先前关于其他小β-折叠铜氧化还原蛋白的研究相比，总体结果支持这样的假设：α-螺旋的存在可以将天青蛋白的折叠机制从分层机制转变为非分层机制，其中高度保守的β-折叠核心为野生型蛋白质的协同折叠提供了支架。
