Leite John F, Cascio Michael
Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Biochemistry. 2002 May 14;41(19):6140-8. doi: 10.1021/bi015895m.
Tetranitromethane (TNM), a small aqueous reagent that specifically modifies solvent-accessible tyrosine residues to o-nitrotyrosine, was used to probe the topology of the GlyR. Homomers of human alpha1 GlyR were recombinantly expressed via a baculovirus system, affinity-purified, and reconstituted in lipid vesicles of defined composition. The native-like reconstituted receptors were then reacted with TNM, and GlyR reaction products were isolated by SDS-PAGE. After proteolytic digestion, TNM-labeled residues were identified using mass spectrometry by observing the mass shift corresponding to the nitrate moiety. In this manner, we have identified TNM modifications of tyrosine residues at positions 24, 75, 78, 161, 223, and 228 in the receptor. Of significance, nitrations at Tyr 223 and Tyr 228 occur within the first putative transmembrane helix (M1) of the receptor, and their labeling suggests a non-helical secondary structure for M1 for the glycine receptor. In a previously published report [Leite et al. (2000) J. Biol. Chem. 275, 13683], we also identified proteolytic cleavage sites within M1. Taken together, these studies support a topological model where the "historical" M1 segment cannot be entirely alpha-helical and may contain an extramembranous surface loop. Furthermore, we have also identified a tyrosine modification (Tyr 161) within a region of the N-terminal domain critical in agonist and antagonist binding.
四硝基甲烷（TNM）是一种小分子水性试剂，它能将溶剂可及的酪氨酸残基特异性地修饰为邻硝基酪氨酸，被用于探究甘氨酸受体（GlyR）的拓扑结构。人α1甘氨酸受体的同聚体通过杆状病毒系统进行重组表达、亲和纯化，并重构于特定组成的脂质囊泡中。然后将具有天然样结构的重构受体与TNM反应，通过SDS-PAGE分离甘氨酸受体反应产物。经过蛋白酶消化后，通过观察对应于硝酸盐部分的质量位移，利用质谱法鉴定TNM标记的残基。通过这种方式，我们确定了受体中第24、75、78、161、223和228位酪氨酸残基的TNM修饰。重要的是，Tyr 223和Tyr 228的硝化发生在受体的第一个假定跨膜螺旋（M1）内，它们的标记表明甘氨酸受体的M1具有非螺旋二级结构。在之前发表的一篇报告中[Leite等人（2000年）《生物化学杂志》275卷，13683页]，我们还确定了M1内的蛋白酶切割位点。综上所述，这些研究支持了一个拓扑模型，即“传统的”M1片段不可能完全是α螺旋，可能包含一个膜外表面环。此外，我们还在对激动剂和拮抗剂结合至关重要的N端结构域区域内确定了一个酪氨酸修饰（Tyr 161）。
