Bodenreider Christophe, Kellershohn Nicolas, Goldberg Michel E, Méjean Annick
Unité de Repliement et Modélisation des Protéines, CNRS URA 2185, Institut Pasteur, 28 rue du Dr. Roux 75724 Paris Cédex 15, France.
Biochemistry. 2002 Dec 17;41(50):14988-99. doi: 10.1021/bi020453b.
R67 dihydrofolate reductase (DHFR) is a homotetrameric enzyme. Its subunit has a core structure consisting of five antiparallel beta-strands that form a compact beta-barrel. Our interest was to describe the molecular mechanism of the complete folding pathway of this beta-sheet protein, focusing on how the oligomerization steps are coordinated with the formation of secondary and tertiary structures all along the folding process. The folding kinetics of R67 dihydrofolate reductase into dimers at pH 5.0 were first examined by intrinsic tryptophan fluorescence, fluorescence energy transfer, and circular dichroism spectroscopy. The process was shown to consist of at least four steps, including a burst, a rapid, a medium, and a slow phase. Measurements of the ellipticity at 222 nm indicated that about 50% of the total change associated with refolding occurred during the 4 ms dead time of the stopped-flow instrument, indicating a substantial burst of secondary structure. The bimolecular association step was detected using fluorescence energy transfer and corresponded to the rapid phase. The slow phase was attributed to a rate-limiting isomerization of peptidyl-prolyl bonds involving 15% of the unfolded population. A complete folding pathway from the unfolded monomer to the native tetramer was proposed and an original model based upon the existence of early partially folded monomeric intermediates, rapidly stabilized in a dimeric form able to self-associate into the native homotetramer was formulated. The rate constants of these various steps were determined by fitting the kinetic traces to this model and supported our mechanistic assumptions.
R67二氢叶酸还原酶（DHFR）是一种同四聚体酶。其亚基具有由五条反平行β链组成的核心结构，这些β链形成一个紧密的β桶。我们感兴趣的是描述这种β折叠蛋白完整折叠途径的分子机制，重点关注在整个折叠过程中寡聚化步骤如何与二级和三级结构的形成相协调。首先通过内在色氨酸荧光、荧光能量转移和圆二色光谱研究了R67二氢叶酸还原酶在pH 5.0时折叠成二聚体的动力学。结果表明该过程至少包括四个步骤，即一个爆发阶段、一个快速阶段、一个中等阶段和一个缓慢阶段。在222 nm处的椭圆率测量表明，与重折叠相关的总变化中约50%发生在停流仪器的4 ms死时间内，这表明二级结构有大量的爆发。使用荧光能量转移检测到双分子缔合步骤，它对应于快速阶段。缓慢阶段归因于涉及15%未折叠群体的肽基脯氨酰键的限速异构化。提出了从未折叠单体到天然四聚体的完整折叠途径，并构建了一个基于早期部分折叠单体中间体存在的原始模型，这些中间体迅速稳定为能够自缔合形成天然同四聚体的二聚体形式。通过将动力学轨迹拟合到该模型确定了这些不同步骤的速率常数，支持了我们的机理假设。
