Department of Chemistry, University of Toronto, Mississauga, Ontario L5L 1C6, Canada.
Biochemistry. 2013 Aug 27;52(34):5780-9. doi: 10.1021/bi4010057. Epub 2013 Aug 16.
Although many proteins are recognized to undergo folding via an intermediate, the microscopic nature of folding intermediates is less understood. In this study, ¹⁹F NMR and near-UV circular dichroism (CD) are used to characterize a transition to a thermal folding intermediate of calmodulin, a water-soluble protein, which is biosynthetically enriched with 3-fluorophenylalanine (3F-Phe). ¹⁹F NMR solvent isotope shifts, resulting from replacing H₂O with D₂O, and paramagnetic shifts arising from dissolved O₂ are used to monitor changes in the water accessibility and hydrophobicity of the protein interior as the protein progresses from a native state to an unfolded state along a heat-denaturation pathway. In comparison to the native state, the solvent isotope shifts reveal the decreased presence of water in the hydrophobic core, whereas the paramagnetic shifts show the increased hydrophobicity of this folding intermediate. ¹⁵N, ¹H and methyl ¹³C,¹H HSQC NMR spectra demonstrate that this folding intermediate retains a near-native tertiary structure whose hydrophobic interior is highly dynamic. ¹⁹F NMR CPMG relaxation dispersion measurements suggest the near-native state is transiently adopted well below the temperature associated with its onset.
虽然许多蛋白质被认为通过中间态进行折叠，但折叠中间态的微观性质还不太清楚。在这项研究中，¹⁹F NMR 和近紫外圆二色性（CD）用于表征钙调蛋白（一种水溶性蛋白质）热折叠中间态的转变，该蛋白质生物合成时富含 3-氟苯丙氨酸（3F-Phe）。¹⁹F NMR 溶剂同位素位移，通过用 D₂O 取代 H₂O 产生，以及溶解氧产生的顺磁位移，用于监测蛋白质从天然状态到展开状态沿着热变性途径进行时，蛋白质内部的水可及性和疏水性的变化。与天然状态相比，溶剂同位素位移表明疏水区中存在的水减少，而顺磁位移表明该折叠中间态的疏水性增加。¹⁵N、¹H 和甲基 ¹³C、¹H HSQC NMR 谱表明，该折叠中间态保留了近乎天然的三级结构，其疏水性内部具有高度动态性。¹⁹F NMR CPMG 弛豫色散测量表明，近天然状态在与其起始相关的温度以下短暂出现。
