Miyoshi Daisuke, Nakao Akihiro, Sugimoto Naoki
Department of Chemistry, Faculty of Science and Engineering, and High Technology Research Center, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan.
Biochemistry. 2002 Dec 17;41(50):15017-24. doi: 10.1021/bi020412f.
Almost all biochemical reactions in vitro have been investigated through numerous experiments conducted in dilute solutions containing low concentrations of solutes. However, biomacromolecules such as nucleic acids, proteins, and polysaccharides are designed to function and/or form their native structures in a living cell containing high concentrations of biomacromolecules, substrates, cofactors, salts, and so on. In the present study, we have demonstrated quantitatively the effect of molecular crowding on structures and stabilities of the G-quadruplex of d(G(4)T(4)G(4)). Molecular crowding with poly(ethylene glycol) (PEG) induced a structural transition from the antiparallel to the parallel G-quadruplex of d(G(4)T(4)G(4)), while molecular crowding with polycations did not alter the structure of the antiparallel G-quadruplex. The binding constants of putrescine, one of the polycations, for d(G(4)T(4)G(4)) in the absence and presence of Na(+) are calculated to be 277 and 2.5 M(-)(1), respectively. This indicates that the polycations coordinate to d(G(4)T(4)G(4)) with electrostatic interactions. The thermodynamic parameters of the antiparallel G-quadruplex formation under the crowding and noncrowding conditions induced by putrescine were also estimated. The stability of the antiparallel G-quadruplex decreased (-DeltaG degrees (25) decreased from 28 to 22 kcal mol(-)(1)) with molecular crowding by putrescine. Also, enthalpy and entropy changes in the structural formation under crowding and noncrowding conditions clearly showed that destabilization was entropy-driven. These quantitative parameters indicated that both the volume excluded by PEG and chemical interactions such as electrostatic interaction with solute polycations are critical for determining how molecular crowding affects the structure and stability of highly ordered DNA structures.
几乎所有体外生化反应都是通过在含有低浓度溶质的稀溶液中进行的大量实验来研究的。然而，核酸、蛋白质和多糖等生物大分子是被设计在含有高浓度生物大分子、底物、辅因子、盐等的活细胞中发挥功能和/或形成其天然结构的。在本研究中，我们定量地证明了分子拥挤对d(G(4)T(4)G(4))的G-四链体结构和稳定性的影响。聚乙二醇（PEG）引起的分子拥挤诱导了d(G(4)T(4)G(4))从反平行G-四链体到平行G-四链体的结构转变，而聚阳离子引起的分子拥挤并未改变反平行G-四链体的结构。计算得出，在不存在和存在Na(+)的情况下，聚阳离子腐胺与d(G(4)T(4)G(4))的结合常数分别为277和2.5 M(-)(1)。这表明聚阳离子通过静电相互作用与d(G(4)T(4)G(4))配位。还估算了腐胺在拥挤和非拥挤条件下诱导反平行G-四链体形成的热力学参数。腐胺引起的分子拥挤使反平行G-四链体的稳定性降低（-ΔG°(25)从28 kcal mol(-)(1)降至22 kcal mol(-)(1)）。此外，拥挤和非拥挤条件下结构形成过程中的焓变和熵变清楚地表明去稳定化是由熵驱动的。这些定量参数表明，PEG排除的体积以及与溶质聚阳离子的静电相互作用等化学相互作用对于确定分子拥挤如何影响高度有序的DNA结构的结构和稳定性至关重要。
