Department of Chemistry, University of Calcutta, Kolkata 700009, India.
Department of Chemistry, University of Calcutta, Kolkata 700009, India.
J Colloid Interface Sci. 2017 Nov 1;505:673-681. doi: 10.1016/j.jcis.2017.06.051. Epub 2017 Jun 19.
The interaction of Ribonuclease A (RNase A) with the bile salt, sodium deoxycholate (NaDC) is meticulously investigated using various spectroscopic techniques. The binding isotherm constructed from the modulation of intrinsic tyrosine fluorescence of the protein following interaction with NaDC conspicuously reveals an intrinsically complex stepwise interaction process which proceeds through the formation of distinct conformational states of the protein. The conformational transitions are found to occur at c=2.2mM, and c=7.2mM of NaDC. These results are subsequently corroborated from the studies of excited-state relaxation dynamics of the intrinsic tyrosine residues of RNase A, and the modulations in fluorescence behavior of an extrinsic probe (ANS) bound to the protein. The far-UV circular dichroism spectral analyses unveil only nominal influence on the secondary structural element of the protein up to [NaDC]=c=2.2mM, which is then followed by marked disruption of the secondary structure of RNase A following further addition of NaDC. This clearly accounts for differential interaction behaviors of RNase A with the monomeric and micellar forms of NaDC (CMC of NaDC in aqueous buffer is estimated to be ∼3.0mM). In Region-I (up to c=2.2mM), the protein:surfactant interaction is argued to be predominantly governed by electrostatic/ionic interaction force. Subsequently, in Region-II (up to c=7.2mM) the RNase A:NaDC interaction accompanies major denaturation of the protein (∼17% loss of the secondary structure of RNase A at c=7.2mM) resulting in significant exposure of hydrophobic surfaces of the protein. However, the tertiary structure of the protein remains essentially unperturbed within the concentration regime of NaDC under study.
使用各种光谱技术，细致地研究了核糖核酸酶 A（RNase A）与胆汁盐脱氧胆酸钠（NaDC）的相互作用。通过与 NaDC 相互作用后蛋白质内源性酪氨酸荧光的调制构建的结合等温线，明显揭示了一个复杂的逐步相互作用过程，该过程通过蛋白质的不同构象状态的形成来进行。发现构象转变发生在 c=2.2mM 和 c=7.2mM 的 NaDC 处。这些结果随后从 RNase A 内源性酪氨酸残基的激发态弛豫动力学研究以及与蛋白质结合的外源性探针（ANS）的荧光行为的调制中得到证实。远紫外圆二色光谱分析仅揭示了在 [NaDC]=c=2.2mM 之前对蛋白质二级结构元件的名义影响，随后随着 NaDC 的进一步添加，RNase A 的二级结构明显被破坏。这清楚地说明了 RNase A 与 NaDC 的单体和胶束形式（在水性缓冲液中 NaDC 的 CMC 估计约为 3.0mM）的相互作用行为存在差异。在区域 I（高达 c=2.2mM），蛋白质-表面活性剂相互作用主要由静电/离子相互作用力控制。随后，在区域 II（高达 c=7.2mM），RNase A:NaDC 相互作用伴随着蛋白质的主要变性（在 c=7.2mM 时 RNase A 的二级结构损失约 17%），导致蛋白质的疏水性表面显著暴露。然而，在研究的 NaDC 浓度范围内，蛋白质的三级结构基本保持不变。
