Cramer J A, Marchesi V T, Armitage I M
Biochim Biophys Acta. 1980 Jan 25;595(2):235-43. doi: 10.1016/0005-2736(80)90086-3.
High-field (270 MHz) 1H-NMR has been employed to study the solution conformation of glycophorin A, a sialoglycoprotein which spans the human erythrocyte membrane. Glycophorin A is one of the most fully characterized integral membrane proteins known, making it an excellent model for the study of membrane-bound proteins. This protein consists of three distinct domains: a glycosylated extracellular N-terminus, a hydrophobic intramembranous segment, and a polar cytoplasmic C-terminus. These domains contain aromatic residues which serve as convenient 1H-NMR conformational probes. The aromatic region of the NMR spectrum of glycophorin A in 2H2O shows single, well-resolved His and Tyr resonances. No resonances are observed, however, for the Phe residues which are located in or near the hydrophobic domain. These observations suggest that considerable heterogeneity with respect to segmental motions exists within the protein. This is consistent with circular dichroism data showing the intramembranous segment to be completely helical with the extremities of the protein being predominantly random coils. The helix of the hydrophrobic domain is remarkably resistant to conventional denaturing conditions including variations in pH, and temperature, and treatment with guanidine hydrochloride. However, in trifluoroacetic acid, which strongly solvates peptide backbones, there is extensive reversible unfolding of the helical structure as evidenced by the appearance of Phe resonances. Solvent titration experiments indicate that approximately a 1 : 1 volume ratio of trifluoroacetic acid to 2H2O is required to initiate unfolding of the helix.
高场（270兆赫）1H-核磁共振已被用于研究血型糖蛋白A的溶液构象，血型糖蛋白A是一种跨越人类红细胞膜的唾液酸糖蛋白。血型糖蛋白A是已知的最具特征的整合膜蛋白之一，使其成为研究膜结合蛋白的优秀模型。这种蛋白质由三个不同的结构域组成：一个糖基化的细胞外N端、一个疏水的膜内片段和一个极性的细胞质C端。这些结构域含有芳香族残基，可作为方便的1H-核磁共振构象探针。在重水（2H2O）中血型糖蛋白A的核磁共振光谱的芳香区显示出单一的、分辨率良好的组氨酸（His）和酪氨酸（Tyr）共振信号。然而，位于疏水结构域内或附近的苯丙氨酸（Phe）残基未观察到共振信号。这些观察结果表明，该蛋白质内部的片段运动存在相当大的异质性。这与圆二色性数据一致，该数据显示膜内片段完全呈螺旋状，而蛋白质的末端主要是无规卷曲。疏水结构域的螺旋对包括pH值、温度变化以及盐酸胍处理在内的传统变性条件具有显著抗性。然而，在强烈溶剂化肽主链的三氟乙酸中，螺旋结构会发生广泛的可逆展开，这可通过苯丙氨酸共振信号的出现得到证明。溶剂滴定实验表明，大约需要三氟乙酸与重水1：1的体积比才能引发螺旋结构的展开。
