Specific monovalent cation effects on the domain-domain interaction of heterogeneous dimeric protein were investigated using green fluorescent protein (GFP)-glutathione-s-transferase (GST) fusion protein as a model protein. Conjugating N-terminal of GST domain with a fluorescence probe Cyanine3, complementary increase and decrease of fluorescence intensities of Cyanine3 and GFP were recognized on the exclusive excitation of GFP and further the fluorescence decay of GFP was remarkably accelerated to show that an excellent Förster type of resonance excitation energy transfer (FRET) pair was constructed between GFP- and GST-domain. The spectral overlap integral and critical distance of the FRET pair were estimated to be 5.96×10¹³ M⁻¹cm³ and 62.5 Å, respectively. The FRET rate and efficiency evaluated by fluorescence lifetime of the energy donor, GFP, were influenced by the monovalent cations included in the buffer solution to suggest that the domain-domain interactions of GFP-GST fusion protein would be susceptible to cation species and their concentrations. The order affecting the domain-domain interaction was estimated to be Li⁺>NH₄⁺ >Na⁺>K⁺>Cs⁺, almost corresponding to the reverse Hofmeister series.

中文翻译：

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通过 FRET 分析揭示的阳离子对异源二聚体蛋白域-域相互作用的特异性影响。

使用绿色荧光蛋白 (GFP)-谷胱甘肽-s-转移酶 (GST) 融合蛋白作为模型蛋白，研究了异质二聚体蛋白域-域相互作用的特定单价阳离子效应。将 GST 结构域的 N 端与荧光探针 Cyanine3 结合，在 GFP 的独家激发下识别出 Cyanine3 和 GFP 荧光强度的互补增加和减少，并且 GFP 的荧光衰减进一步显着加速，表明优异的 Förster 型在 GFP 和 GST 域之间构建了共振激发能量转移 (FRET) 对。FRET 对的光谱重叠积分和临界距离估计为 5.96×10 ¹³ M ⁻¹ cm ³和 62.5 Å，分别。通过能量供体 GFP 的荧光寿命评估的 FRET 速率和效率受到缓冲溶液中包含的单价阳离子的影响，表明 GFP-GST 融合蛋白的域-域相互作用易受阳离子种类及其浓度的影响. 影响域-域相互作用的顺序估计为Li ⁺ >NH ₄ ⁺ >Na ⁺ >K ⁺ >Cs ⁺，几乎对应于逆霍夫迈斯特级数。