Gloss L M, Matthews C R
Department of Chemistry, Center for Biomolecular Structure and Function, Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1998 Nov 10;37(45):15990-9. doi: 10.1021/bi981511p.
A polypeptide corresponding to the core/dimerization domain of E. coli Trp repressor (TR), [2-66]2 TR, was constructed by insertion of a pair of stop codons into the trpR gene. The kinetic properties of the urea-induced folding of this core fragment were examined by intrinsic tryptophan fluorescence (FL) and circular dichroism (CD) spectroscopy. The kinetic response of wild-type TR (WT TR) is very complicated and has been interpreted to involve three parallel channels with multiple folding and isomerization reactions (Mann et al. (1995) Biochemistry 34, 14573-14580). The refolding of [2-66]2 TR can be described by a much simpler mechanism, involving an association reaction followed by a urea-dependent first-order folding reaction. The second-order rate constant for the association reaction approaches that of the diffusion limit, 3 x 10(8) M-1 s-1 in 1 M urea at 15 degreesC. Double-jump experiments demonstrate that >/=93% of the unfolded monomers proceed to the native dimer via the dimeric intermediate; several lines of evidence demonstrate that this dimeric species is an on-pathway intermediate. The subsequent first-order folding reaction of the dimeric intermediate to the native species involves development of additional secondary structure and tertiary structure. The kinetic folding mechanism of [2-66]2 TR suggests that: (1) the complexity of the folding kinetics of full-length WT TR arises from alternative interactions of the DNA reading heads with the dimerization core domain-not from the intertwined nature of the dimerization interface; (2) residues 2-66 contain all of the sequence information necessary to direct the near-diffusion-limited association reaction in a TR folding reaction; and (3) the formation of secondary and tertiary structure is concurrent with or precedes dimerization, and further development certainly follows the formation of quaternary structure.
通过在trpR基因中插入一对终止密码子，构建了一种与大肠杆菌色氨酸阻遏蛋白（TR）的核心/二聚化结构域相对应的多肽，即[2-66]2 TR。通过内源色氨酸荧光（FL）和圆二色性（CD）光谱研究了该核心片段尿素诱导折叠的动力学特性。野生型TR（WT TR）的动力学响应非常复杂，已被解释为涉及三个具有多个折叠和异构化反应的平行通道（Mann等人，（1995年）《生物化学》34卷，14573 - 14580页）。[2-66]2 TR的重折叠可以用一个简单得多的机制来描述，包括一个缔合反应，随后是一个依赖尿素的一级折叠反应。在15℃的1 M尿素中，缔合反应的二级速率常数接近扩散极限，为3×10^8 M^-1 s^-1。双跳跃实验表明，≥93%的未折叠单体通过二聚体中间体形成天然二聚体；多条证据表明这种二聚体物种是一个折叠途径中的中间体。二聚体中间体随后向天然物种的一级折叠反应涉及额外二级结构和三级结构的形成。[2-66]2 TR的动力学折叠机制表明：（1）全长WT TR折叠动力学的复杂性源于DNA读头与二聚化核心结构域的交替相互作用，而非二聚化界面的缠绕性质；（2）2 - 66位残基包含了在TR折叠反应中指导近扩散极限缔合反应所需的所有序列信息；（3）二级和三级结构的形成与二聚化同时发生或先于二聚化，而进一步的发展肯定在四级结构形成之后。
