Vidusek D A, Roberts M F, Goodfriend L
Biochemistry. 1985 May 21;24(11):2747-53. doi: 10.1021/bi00332a023.
The solution conformation of short ragweed allergen Ra5, a protein of 45 amino acid residues cross-linked with four disulfide bridges, has been investigated by 1H NMR spectroscopy at 500 MHz. The aromatic region, which contains resonances from three tyrosines and two tryptophans, has been partially assigned. Two tyrosines titrate with a pK of 10.2; a third tyrosine is buried under the tryptophan resonances, and its pK could not be determined. The two tryptophans reside in different microenvironments; the resonances of one are very similar to those found in random coil structures while the other has dramatically shifted peaks. Nuclear Overhauser effect (NOE) difference spectroscopy is used to define two distinct spin-diffusion systems for the aromatic residues and to further identify several methyl-containing amino acids involved in these systems. Assignments in the methyl region are based on selective decoupling, chemical shifts, NOE difference spectra, and 2-D J-resolved and 2-D J-correlated spectroscopy (COSY) methodology. A unique ring-current-shifted methyl doublet in the Ra5 spectrum titrates into the bulk methyl region with a pK of 10.2. Examination of the COSY map suggests that this resonance belongs to either leucine-1 or isoleucine-38. Chemical removal of the N-terminal leucine did not affect the ring-current-shifted methyl. Therefore, this unique resonance has been assigned to the methyl of isoleucine-38.(ABSTRACT TRUNCATED AT 250 WORDS)
已通过500兆赫的1H核磁共振光谱法研究了短豚草过敏原Ra5的溶液构象，该蛋白由45个氨基酸残基组成，有四个二硫键交联。包含三个酪氨酸和两个色氨酸共振信号的芳香区已部分完成归属。两个酪氨酸的滴定pK值为10.2；第三个酪氨酸埋藏在色氨酸共振信号之下，其pK值无法确定。两个色氨酸处于不同的微环境中；其中一个的共振信号与无规卷曲结构中的非常相似，而另一个的峰则有显著位移。核Overhauser效应（NOE）差光谱法用于定义芳香族残基的两个不同自旋扩散系统，并进一步鉴定参与这些系统的几个含甲基氨基酸。甲基区的归属基于选择性去耦、化学位移、NOE差光谱以及二维J分辨和二维J相关光谱（COSY）方法。Ra5光谱中一个独特的环电流位移甲基双峰以10.2的pK值滴定到大量甲基区。对COSY图谱的检查表明，该共振信号属于亮氨酸-1或异亮氨酸-38。化学去除N端亮氨酸不影响环电流位移甲基。因此，这个独特的共振信号已归属为异亮氨酸-38的甲基。（摘要截短于250字）
