Daniel W E, Cohn M
Proc Natl Acad Sci U S A. 1975 Jul;72(7):2582-6. doi: 10.1073/pnas.72.7.2582.
Thiouridine at position 8 (s4U8) of tRNAf1Met was spin-labeled with the nitroxide free radical, N-(1-oxyl-2,2,5,5-Tetramethyl-3-pyrrolidinyl) bromacetamide, for proton nuclear magnetic resonance spectroscopic studies. The well-resolved methyl peak of ribothymidine is unperturbed, but the peak tentatively assigned to the C-5 methylene group of dihydrouridine is considerably broadened in spin-labeled tRNAf1Met. Of the approximately 27 slowly exchanging protons observed in the region between 11 and 15 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid, the equivalent of about five protons apparently disappeared in spin-labeled tRNAf1Met. The well-resolved single proton at 14.8 ppm was missing not only in the paramagnetic species, but also in the diamagnetic reduced form of spin-labeled tRNAf1Met, and was unequivocally identified as a hydrogen bond involving s4U8 by comparison of several forms of tRNAf1Met specifically modified at s4U. Evidence that the perturbation of a second single proton resonance at 14.6 ppm (shift and broadening) is coupled to the loss of a tertiary hydrogen bond involving residue 8, arises from the same modified forms. The resolved resonances in the methyl and N-H regions, particularly the resonance at 14.6 ppm as well as the four N-bonded proton resonances at higher field which are broadened solely due to their proximity to the unpaired electron of the spin label, provide specific indicators of the geometry of tRNAf1Met structure in solution. Their observability by nuclear magnetic resonance spectroscopy opens up the possibility of monitoring distance changes among the base residues of spin-labeled tRNAf1Met upon its interaction with aminoacyl-tRNA synthetase and other enzymes.
对tRNAf1Met第8位的硫代尿苷（s4U8）用氮氧自由基N-(1-氧代-2,2,5,5-四甲基-3-吡咯烷基)溴乙酰胺进行自旋标记，用于质子核磁共振光谱研究。核糖胸腺嘧啶的甲基峰解析良好且未受干扰，但在自旋标记的tRNAf1Met中，初步归属于二氢尿苷C-5亚甲基的峰显著变宽。在相对于4,4-二甲基-4-硅戊烷-1-磺酸的场强下11至15 ppm区域观察到约27个缓慢交换的质子，在自旋标记的tRNAf1Met中约五个质子的等效物明显消失。在14.8 ppm处解析良好的单质子不仅在顺磁物种中缺失，在自旋标记的tRNAf1Met的抗磁还原形式中也缺失，通过比较几种在s4U处特异性修饰的tRNAf1Met形式，明确鉴定其为涉及s4U8的氢键。来自相同修饰形式的证据表明，在14.6 ppm处第二个单质子共振的扰动（位移和变宽）与涉及第8位残基的三级氢键的丧失相关。甲基和N-H区域中解析的共振，特别是14.6 ppm处的共振以及高场处仅由于靠近自旋标记的未成对电子而变宽的四个N键合质子共振，提供了溶液中tRNAf1Met结构几何形状的特定指标。它们通过核磁共振光谱的可观测性为监测自旋标记的tRNAf1Met与氨酰-tRNA合成酶和其他酶相互作用时碱基残基之间的距离变化开辟了可能性。
